Deleted adenovirus vectors and methods of making and administering the same

ABSTRACT

The present invention provides deleted adenovirus vectors. The inventive adenovirus vectors carry one or more deletions in the IVa2, 100K, polymerase and/or preterminal protein sequences of the adenovirus genome. The adenoviruses may additionally contain other deletions, mutations or other modifications as well. In particular preferred embodiments, the adenovirus genome is multiply deleted, i.e., carries two or more deletions therein. The deleted adenoviruses of the invention are “propagation-defective” in that the virus cannot replicate and produce new virions in the absence of complementing function(s). Preferred adenovirus vectors of the invention carry a heterologous nucleotide sequence encoding a protein or peptide associated with a metabolic disorder, more preferably a protein or peptide associated with a lysosomal or glycogen storage disease, most preferably, a lysosomal acid α-glucosidase. Further provided are methods for producing the inventive deleted adenovirus vectors. Further provided are methods of administering the deleted adenovirus vectors to a cell in vitro or in vivo.

RELATED APPLICATION INFORMATION

This application claims the benefit of provisional application Ser. No.60/145,742 filed on Aug. 28, 1998, which is incorporated herein byreference in its entirety.

FEDERAL SUPPORT

Government support for this invention was provided by Grant NumberDK52925-02 from the National Institutes of Health. The government hascertain rights to this invention.

FIELD OF THE INVENTION

The present invention relates to adenovirus vectors, more particularly,to propagation-defective adenovirus vectors.

BACKGROUND OF THE INVENTION

The basis of gene therapy is to deliver a functional gene to tissueswhere the respective gene activity is missing or defective. Among theapproaches to accomplishing gene therapy has been the use of recombinantviral vectors which have been genetically engineered to carry a desiredtransgene. These viral-based vectors have advantageous characteristics,such as the natural ability to infect the target tissue. However,implementation of existing viral vectors are impeded by severallimitations as well.

For example, retrovirus-based vectors must integrate into the genome ofthe target tissue to allow for transgene expression (with the potentialto activate resident oncogenes) while vector titers produced in suchsystems are significantly less than in some other systems. Because ofthe requirement for integration into the subject genome, the retrovirusvector can only be used to transduce actively dividing tissues. Further,many retroviruses have limited host tissue specificity and cannot beemployed to transduce more than a few specific tissues of the subject.

Adenovirus vectors hold great promise for gene therapy. Adenovirusvectors can transduce multiple types of tissues in vivo, includingnondividing, differentiated cells such as those found in liver, kidney,muscle (skeletal and cardiac), respiratory, and nervous system tissues.See, e.g., Askari et al., Gene Ther. 3:381-388 (1996); Barr et al., GeneTher. 1:51-58 (1994); Engelhardt et al., Hum. Gene Ther. 4:759-769(1993). Using transcomplementing packaging cell lines, first generationAdenovirus vectors can be grown and concentrated to high titers (>10¹³),which contributes to their ability to transduce large numbers of targetcells after in vivo administration. Ragot, et al., Nature 361:647-50(1993).

First generation adenovirus vectors also have a comparatively largecarrying capacity (i.e., up to about 8.0 kb). The ability of firstgeneration Adenovirus vectors to allow expression of transduced genesepisomally for extended periods of time in immune-incompetent andsometimes immune-competent animals without the need for integration intothe vector genome (Vincent, et al., Nat Genet: 5:130-34 (1993);Tripathy, et al., Nature Medicine 2:545-50 (1996)) allows them totransduce mitotically quiescent cells as well as actively dividingcells. Finally, live Adenovirus preparations have been used for thevaccination of military recruits, and Ad strains 2 and 5 (most commonlyused for vector development) are not associated with severe disease.

Despite the advantages discussed above, first generation, E1-deletedadenovirus virus vectors are limited in potential therapeutic use forseveral reasons. First, due to the size of the E1 deletion and tophysical virus packaging constraints, first generation adenovirusvectors are limited to carrying approximately 8.0 kb of transgenegenetic material. While this compares favorably with other viral vectorsystems, it limits the usefulness of the vector where a larger transgeneis required. Second, infection of the E1-deleted first generation vectorinto packaging cell lines leads to the generation of some replicationcompetent adenovirus particles, because only a single recombinationevent between the E1 sequences resident in the packaging cell line andthe adenovirus vector genome can generate a wild-type virus. Therefore,first-generation adenovirus vectors pose a significant threat ofcontamination of the adenovirus vector stocks with significantquantities of replication competent wild-type virus particles, which mayresult in toxic side effects if administered to a gene therapy subject.

The most difficult problem with adenovirus vectors is their inability tosustain long-term transgene expression, secondary to hose immuneresponses that eliminate virally transduced cells in immune-competentanimals. Gilgenkrantz et al., Hum. Gene Ther. 6:1265-1274 (1995); Yanget al., J. Virol. 69:2004-2015 (1995); Yang et al., Proc. Natl. Acad.Sci. USA 91:44074411 (1994); Yang et al., J. Immunol. 155: 2565-2570(1995). While immune responses have been demonstrated against thetransgene-encoded protein product (Tripathy et al., Nat. Med. 2; 545-550(1996)), it has also been demonstrated that adenovirus vector epitopesare major factors in triggering the host immune response. Gilgenkrantzet al., Hum. Gene Ther. 6:1265-1274 (1995); Yang et al., J. Virol. 70:7209-7212 (1996). It has been repeatedly demonstrated that transgenesuch as the bacterial β-galactosidase gene are highly immunogenic whentransduced by adenovirus vectors, in contrast to other delivery systems(e.g., direct DNA injection or adeno-associated virus administration),where an immune response against the immunogenic transgene is lackingand transgene expression persists. Woff et al., Hum. Mol. Genet.1:363-369 (1992); Xiao et al., J. Virol. 70:8098-8108 (1996).

In addition, E1⁻ vectors have also been reported to express theadenovirus early genes, undergo genome replication and express the L1-L5encoded structural genes when utilized in vivo. E. g., Yang, et al.,Immunity 1: 433-442 (1994). Because only a single recombination event isrequired to produce an entirely replication competent virus from the E-1deletion, the exaggerated immune response may also be due in someinstances to the contaminating presence of wild type adenovirus virus inthe vector preparation. Eg., Rich, Hum. Gene. Ther. 4: 461-476 (1993).Either (or both) of these phenomena result in the production andpresence of viral proteins in the transduced cells, possibly creating ahigher antigenic profile than other gene therapy vector systems. Thepresence of these adenovirus viral gene products may contribute to theshort duration of transgene expression in cells infected by firstgeneration adenovirus vectors by accelerating the detection andelimination of adenovirus vector infected cells by the host immunesystem.

Accordingly, there remains a need in the art for improved adenovirusvector systems that address the limitations of existing systems.

SUMMARY OF THE INVENTION

The present invention provides novel deleted adenovirus vectors thatprovide advantages over existing “first-generation” adenovirus vectors.The deleted adenovirus vectors of the present invention mayadvantageously have an increased carrying capacity for heterologousnucleotide sequences, demonstrate lower levels of viral proteinexpression, induce fewer host immune responses, and/or exhibit increasedstability and prolonged transgene expression when introduced into targetcells.

The inventive adenovirus vectors carry one or more deletions in theIVa2, 100K, polymerase and/or preterminal protein sequences of theadenovirus genome. The adenoviruses may additionally contain otherdeletions, mutations or modifications as well. In particular preferredembodiments, the adenovirus genome is multiply deleted, i.e., carriestwo or more deletions therein. More preferably, there are deletions intwo or more regions of the adenovirus genome (e.g., E1, E3, polymerase,100K, IVa2, preterminal protein, etc.). At least one of the deletions inthe adenovirus genome renders the adenovirus “propagation-defective” inthat the virus cannot replicate and produce new virions in the absenceof complementing function(s); preferably the vector carries multiple(two or more) deletions that result in a propagation-defectivephenotype. When introduced into a trans-complementing cell that providesthe deleted functions from the adenovirus genome, the deletedadenoviruses of the invention can produce a productive infection thatresults in the generation of new virus particles.

A further aspect of the present invention is methods for producinghigh-iters of the inventive deleted adenovirus vectors using packagingcells. Methods are also disclosed for producing the inventive deletedvectors using bacterial recombination and methods for producing “gutted”adenovirus vectors using deleted helper adenoviruses. Gutted adenovirusstocks according to the invention may exhibit increased stability andreduced viral protein expression from contaminating helper viruses ascompared with previous preparations.

The inventive vectors can be administered to cells in vitro, e.g., toproduce a proteinipeptide or RNA of interest. In particular, arecombinant deleted adenovirus vector according to the invention may beadministered to a cell in vitro, whereupon the cell expresses aheterologous nucleotide sequence. In particular embodiments, thenucleotide sequence encodes a protein or peptide (e.g., an enzyme) thatis related to a metabolic disorder and/or a lysosomal or glycogenstorage disorder. The expressed protein or peptide can be isolated,e.g., for protein replacement therapies.

In other embodiments, the recombinant adenovirus vectors of theinvention may be administered to a cell in vitro and the celladministered to a subject, e.g., to produce an immunogenic ortherapeutic response in the subject. In further alternative embodiments,the inventive deleted adenovirus vectors are administered directly tothe subject. In particular, the present investigations have determinedthat intravenous administration to GAA-deficient animals of deletedadenovirus vectors of the invention carrying a GAA gene resulted inhigh-level transduction of liver cells and subsequent expression of theGAA transgene. Hepauic expression of GAA produced elevated plasma levelsof GAA protein and significant reductions in glycogen levels in affectedtissues.

The present invention also discloses methods of administering arecombinant deleted adenovirus vector of the invention into an organ ortissue, whereby a heterologous nucleotide sequence is expressed and theencoded proteinipeptide or RNA is delivered to a different organ ortissue, e.g., to produce an immunogenic of therapeutic effect. Forexample, a nucleotide sequence encoding a foreign protein can bedelivered to the liver, whereupon it is expressed and secreted into thecirculatory system and delivered to target tissues (e.g., muscle). As afurther alternative, the inventive adenovirus vectors can be introducedinto the brain (e.g., by direct injection) for delivery of foreignproteins or nucleotide sequences to the central nervous system.

A further aspect of the invention is methods of expressing a protein orpeptide in the liver for delivery to a distal tissue or organ (e.g.,muscle tissue) to provide a therapeutic effect therein. Preferably, adeleted adenovirus of the invention is employed to introduce anucleotide sequence encoding the protein or peptide into the liver. Alsopreferred are nucleotide sequences encoding proteins or peptidesassociated with a metabolic disorder, more preferably a lysosomal orglycogen storage disease (e.g., lysosomal acid α-glucosidase). Furtherpreferred are nucleotide sequences encoding lysosomal proteins orpeptides.

These and other aspects of the invention are set forth in more detail inthe description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagrammatic representation of the steps utilized toisolate respectively Panel A: pBHG11Δpol, Panel B: AdΔpol andAdΔpol/pBHG11, and Panel C: AdLacZΔpol.

FIG. 2 shows AdLacZΔpol DNA replication after infection of 293 cells atMOI of 1.5 BFU at 0, 20 and 48 hours after infection.

FIG. 3 shows AdLacZΔpol DNA replication after infection of B-6 cells atvarious MOIs at 0, 10, 24, and 48 hours after infection.

FIG. 4 shows kinetics of growth of AdLacZΔpol in 293, B-6 and C-7 cells.

FIG. 5 shows kinetics of growth of AdLacZΔpp in cell lines 293, B-6 andC-7 and Adsub360LacZ in line 293.

FIG. 6 shows AdLacZΔpol fiber protein synthesis after infection of 293cells at MOI of 1.5 BFU.

FIG. 7 shows AdLacZΔpol fiber protein synthesis after infection of B-6cells at MOI of 1.5 BFU.

FIG. 8 shows the lack of expression of the adenovirus late gene product,fiber protein, in HeLa cells infected with AdLacZΔpol and AdLacZΔppcompared with Adsub360LacZ.

FIG. 9 shows expression of the lacZ gene in vivo (A) after infection ofBALB/c mice as compared with (B) mock-infected control.

FIG. 10 shows a HindIII restriction enzyme analysis for each of theindicated adenoviruses.

FIG. 11 shows NotI and EcoRI digests of AdLacZΔpol infected B-6 cellscompared with NotI and EcoRI digested dl7001 DNA.

FIG. 12 shows relative growth of AdLacZΔpol when infected at an MOI of0.01 into 293 cells (provide E1) and B-6 cells (provide E1 and pol).

FIG. 13 shows a diagrammatic representation of the steps used to isolateAdLacZΔpp.

FIG. 14 shows restriction enzyme analysis to confirm the genomicstructure of Δpol, ΔpTP (referred to as AdLacZΔpp) and compares it toother adenovirus vectors of known genome structure.

FIG. 15 is a schematic representation of the portion of the adenovirusgenome encoding the polymerase and preterminal protein open-readingframes, and indicating the areas deleted in the AdLacZΔpp vector.

FIG. 16 shows the level of replication in AdLacZΔpp when grown in thepackaging cell line C-7 which provides E1, pol and pTP gene activity ascompared with packaging cell lines 293 (provides only E1) and B-6(provided E1 and pol).

FIG. 17 shows the level of replication of Adsub360LacZ, AdLacZΔpol, orAdLacZΔpp when grown in HeLa cells, which lack E1, polymerase, and pTPactivities. The AdLacZΔpol and AdLacZΔpp genomes persisted 48 hoursafter infection.

FIG. 18 shows expression of the LacZ genes in vivo after introduction ofAdsub360LacZ (Panel B), AdLacZΔpp (Panel C) or AdLacZΔpol (Panel D) ormock infection (Panel A) into 7- to 9- week-old SCID or C57B1/6 immunedeficient mice.

FIG. 19 shows the structure of shuttle plasmid pAdAscL-ΔIVa2.

FIG. 20 shows the results of PCR amplification of K-16 and 293 (Panel A)genomic DNA using primers specific to the 100K region. Panel B showsthat equivalent amounts of amplified DNA were loaded in each lane. PanelC shows a Northern blot analysis of 293 and K-16 total RNA using a 100Kspecific probe.

FIG. 21 shows a Northern blot using a 100K specific probe of RNAisolated from C7 cells constitutively expressing the 100K gene (lanes1-4), as well as a cell line expressing E1 alone (lane 6) or E1 and E1and 100K (land 5).

FIG. 22 shows persistence of LacZ neoantigen expression after AdLacZΔpoltransduction at various days post infection. Each panel represents anindividual animal. Transduction was accomplished with Adsub360LacZ inpanels a, c and g and with AdLacZΔpol in panels b, d, e, f and h.

FIG. 23 shows the quantification of bacterial β-galactosidase levels intransduced hepatic tissues after transduction by first generationadenovirus LacZ vectors and Ad LacZ vectors of the present invention.

FIG. 24 shows β-galactosidase RNA expression in tissues of animalsinfected with Adsub360Lac Z or AdLacZΔpol.

FIG. 25 represents quantification of β-galactosidase RNA expression intissues of animals infected with Adsub360LacZ or AdLacZΔpol.

FIG. 26 shows the greater persistence of the vector genome DNA ofAdLacZΔpol as compared with Adsub360LacZ.

FIG. 27 shows levels of inflammation after hepatic transduction witheither Adsub360LacA or AdLacZΔpol by haemotoxylin and eosin stainedsections. Panels a, b and c are Adsub360LacZ infected mice. Panels d, eand f are from AdLacZΔpp infected mice.

FIG. 28 shows plasma aspartate aminotransferase (AST) analysis ofhepatic cells of the mice which had been administered Adsub360LacZ orAdLacZΔpol vectors, reflecting decreased AST levels in the Δpol vectoras compared to the first generation vector

FIG. 29 shows the construction of the AdhGAAΔpol vector.

FIG. 30 shows hGAA activities after infection of 293 cells withAdhGAAΔpol.

FIG. 31 shows hGAA enzyme levels after AdhGAAΔpol infection of c57Bl/6mice (wild type).

FIG. 32 shows hGAA immunoblot detection of various isoforms of GAA afterinfection of GAA knockout (KO) mice with AdhGAAΔpol.

FIG. 33 shows GAA activity in various tissues of GAA knockout mice 12days after treatment with AdhGAAΔpol.

FIG. 34 shows a Northern blot analysis of RNA isolated from varioustissues from wild-type and GAA-KO animals administered AdhGAAΔpol at 12days d.p.i using a GAA-specific probe.

FIG. 35 shows glycogen levels by PAD staining in various tissues of GAAknockout mice 12 days after treatment of the animals with AdhGAAΔpol.

FIG. 36 shows the structure of shuttle plasmid pAdAscL-ΔIVa2, Δpp-1.6kb.

FIG. 37 shows the structure of shuttle plasmid pAdAscL-ΔIVa2, Δpp-2.4kb.

FIG. 38 shows the structure of shuttle plasmid pAdAscL-ΔIVa2, Δpol.

DETAILED DESCRIPTION OF THE INVENTION

One strategy for overcoming the limitations of the currently availableadenovirus vector systems is to introduce further deletions in essentialgene regions of the adenovirus vector backbone. The addition of furtherdeletions may increase the transgene carrying capacity of the vectorand, if properly chosen, may reduce or eliminate recombination eventswhich cause the contamination of viral stocks with propagation-competentwild type viral particles, thereby rendering these viral preparationsunsuitable for therapeutic purposes.

Use of “second-generation” deleted adenovirus vectors may also addressanother significant problem of adenovirus vectors—the inability tosustain long term expression of transgenes after transfer intoimmune-competent animals, due to host immune responses and/or promotershutdown (Yang et al., (1994) Proc. Natl. Acad. Sci. USA 91:4407; Yanget al., (1995) J. Virol. 69:2004; Yang et al, (1995) J. Immunol.165:2564; Gilgenkranz et al., (1995) Human Gene Therapy 6:1265; Broughet al, (1997) J. Virol. 71:9206). The host immune responses followingtransduction with first-generation E1-deleted adenovirus vectors havebeen demonstrated to be directed against both transgene andadenovirus-vector encoded epitopes (Tripathy et al., (1996) NatureMedicine 2:545; Gilgenkranz et al., (1995) Human Gene Therapy 6:1265;Yang et al. (1996) J. Virol. 70:7209). It appears that first generationadenovirus vectors elicit an exaggerated host immune response. Forexample, adenovirus mediated transfer of the bacterial β-galactosidasegene has been demonstrated to be highly immunogenic in immune-competentanimals, in contrast to other delivery systems such as direct DNAinjection or adeno-associated virus administration, where an immuneresponse to Sgalactosidase has been demonstrated to be conspicuouslylacking (Xiao et al., (1996) J. Virol. 70:8098; Wolff et al., (1996)Hum. Mol. Genet. 1:363).

The deleted adenovirus vectors of the present invention may reduce oreliminate viral DNA replication and/or viral gene product productionwhen infected into the target cells as compared with first-generationE1-deleted vectors. While not wishing to be held to any particulartheory of the invention, it appears that reduced viral replicationadvantageously results in prolonged transgene expression followingtransduction with the deleted adenovirus vectors of the invention.Reduced viral specific activities following vector transduction andtransgene expression may also result in reduced host immune responsesand cytotoxic effects on the host cell. These, in turn, may result in alonger duration of transgene expression.

Use of the vectors of the present invention may also dramatically reducethe host immune response to the transduced cells by offering a moreeffective blockage of viral DNA replication and viral gene expression.This may eliminate or reduce the need for administration of non-specific(and sometimes toxic) immunosuppressive agents in clinicaladenovirus-based gene therapy protocols. While immune responses toadenovirus-based gene therapy vary depending on the background strain ofthe animal tested, the promoter/enhancer elements utilized to driveexpression of the transgene, and the viral backbone itself, the dramaticimprovement seen in the vectors of the present invention maysignificantly improve performance by reducing the immunologic profile ofthe transduced cells over earlier generation vectors regardless of thesefactors.

Moreover, use of the second-generation vectors may increase thetransgene carrying capacity of adenovirus vectors to 9 kb, 11 kb, oreven 12.5 kb or more from the current limit of 8 kb. This increasedcarrying capacity is advantageous when considering the transfer oflarger cDNA minigene constructs (e.g., dystrophin), the utilization oflarger, tissue-specific promoter/enhancer elements (e.g., themuscle-creatine kinase enhancer), and the reintroduction into vectors ofadenovirus genes that may minimize immune recognition of adenovirusinfected cells in vivo (e.g., the E4 gene) (Cox et al., (1993) Nature364:725; Ilan et al., (1997) Proc. Natl. Acad. Sci. USA 94:2587;Kumarsingh et al., (1996) Human Molecular Genetics 5:913).

Further, the introduction of multiple deletions may reduce thelikelihood of contamination of the resulting vector stocks byreplication competent viruses since multiple recombination events wouldbe required to regenerate a viable virus from a multiply-eleted virus.

Surprisingly, the deleted adenovirus vectors of the present inventionare not unstable. This contrasts with the reports that gutted adenovirusvector depend on preterminal protein activity for genomic stability(Lieber et al., (1996) J. Virol. 70:8944; Lieber et al., (1997) NatureBiotech. 15:1383).

I. Deleted Adenovirus Vectors.

The present invention is based, in part, on the discovery of noveladenovirus vectors containing deletions within the adenovirus polymerase(pol), preterminal protein (pTP), 100K, and IVa2 regions of theadenovirus genome. These novel “second-generation” adenovirus vectorsshould be less “leaky” when transduced into target cells (e.g., for genetherapy) and be able to carry larger heterologous nucleotide sequencesthan previously-described E1 deleted adenovirus vectors. In addition,the deleted adenovirus vectors of the present invention may be lessimmunogenic, and thereby less susceptible to immune clearance, than theE1 deleted vectors previously known in the art.

The term “adenovirus” as used herein is intended to encompass alladenoviruses, including the Mastadenovirus and Aviadenovirus genera. Todate, at least forty-seven human serotypes of adenoviruses have beenidentified (see, e.g., FIELDS et al., VIROLOGY, volume 2, chapter 67 (3ded., Lippincont-Raven Publishers). Preferably, the adenovirus is aserogroup C adenovirus, still more preferably the adenovirus is serotype2 (Ad2) or serotype 5 (Ad5).

In one preferred embodiment, the inventive adenoviruses of the presentinvention are infectious, but propagation-defective, i.e., they cannotreplicate and package new virions in the absence oftranscomplementation, e.g., by a packaging cell that expresses thefunctions deleted from the adenovirus genome. Alternatively stated, thepropagation-defective adenoviruses of the invention cannot produce aproductive infection in the absence of transcomplementation. Thepropagation-defective adenovirus particles carry an adenovirus genomethat has one or more deletions in one or more of the 100K, IVa2, and/orpreterminal protein regions. The deletion(s) in the adenovirus genomepreferably prevents, or essentially prevents, the expression of afunctional form of the indicated protein from the deleted region. Forexample, the 100K deletion preferably prevents, or essentially prevents,the expression of a functional 100K protein from the deleted 100K regionof the adenovirus genome. The IVa2 deletion preferably prevents, oressentially prevents, the expression of a functional IVa2 protein fromthe deleted IVa2 region of the adenovirus genome. The preterminalprotein deletion preferably prevents, or essentially prevents, theexpression of a functional preterminal protein from the deletedpreterminal protein region of the adenovirus genome.

By “infectious”, as used herein, it is meant that the adenoviruses canenter the cell by natural transduction mechanisms and express thetransgene therein. Alternatively, an “infectious” adenovirus is one thatcan enter the cell by other mechanisms and express the transgenetherein. As one illustrative example, the vector can enter a target cellby expressing a ligand or binding protein for a cell-surface receptor inthe adenovirus capsid or by using an antibody(ies) directed againstmolecules on the cell-surface followed by internalization of thecomplex, as is described hereinbelow.

In an alternate preferred embodiment, the propagation-defectiveadenovirus includes one or more deletions in the E1 region and one ormore deletions in the polymerase region of the adenovirus genome. Thepolymerase deletion preferably prevents, or essentially prevents, theexpression of a functional polymerase protein from the deletedpolymerase region of the adenovirus genome. The E1 deletion(s)preferably prevents, or essentially prevents, the expression of afunctional form of at least one E1 protein.

In another preferred embodiment, the present invention provides adeleted propagation-defective adenovirus comprising one or moredeletions in the polymerase region of the adenovirus genome. Preferably,the deletion(s) prevents, or essentially prevents, the expression of afunctional polymerase protein from the deleted region. In a furtherpreferred embodiment, the present invention provides deletedpropagation-defective adenoviruses carrying two or more deletions in twoor more regions of the adenovirus genome, wherein one of the deletedregions is in the polymerase region. In particular embodiments whereinthere are two deleted regions and the first deleted region is a singledeletion at nucleotides 7274 to 7881 of the polymerase region, thesecond deleted region is any other region as described herein with theexception of the E3 region. In more preferred embodiments, theadenovirus has one or more deletions in the polymerase region and one ormore deletions in the E1 region, and optionally, one or more deletionsin other regions of the adenovirus genome.

In a further alternative preferred embodiment, the present inventionprovides an infectious, propagation-defective, adenovirus comprising anadenovirus genome containing one or more heterologous nucleotidesequences encoding a lysosomal acid α-glucosidase (GAA), more preferablya human GAA (hGAA), and one or more deletions in one or more of the100K, IVa2, polymerase and/or preterminal protein regions. Thedeletion(s) preferably prevents, or essentially prevents, the expressionof a functional 100K, IVa2, polymerase and/or preterminal protein,respectively, from the deleted region.

The term “prevents the expression” of a functional protein, as usedherein, means that no detectable protein activity is detectable. Thedefect may be at the level of transcription, translation and/orpost-translational processes. Thus, even if there is transcription andtranslation of the deleted gene(s), the resulting protein has nodetectable biological activity. The term “essentially prevents theexpression” of a functional adenovirus protein, as used herein, meansthat only an insignificant amount of biological activity attributable tothe protein is detectable. For example, one way of detecting functionalprotein activity is by monitoring the production of encapsidatedadenovirus. In the presence of a deletion that “essentially prevents theexpression” of a functional adenovirus protein, only an insignificantamount of new adenovirus particles will be produced in the absence ofcomplementation. Packaging of new virions by the inventive deletedadenoviruses, in the absence of complementing functions, is less thanabout 10%, 5%, 2%, 1%, 0.05%, or even 0.01% of the levels detected withwild-type adenoviruses or the deleted adenoviruses in the presence ofcomplementing functions.

The inventive deleted adenoviruses cannot propagate (ie., replicate andpackage new virus particles) without the provision of complementingfunctions to compensate for the deletions(s), e.g., transcomplementationby a packaging cell. As described in more detail hereinbelow, thepackaging cell will typically express and provide the functional proteinthat cannot be expressed from the adenovirus genome as a result of thedeletion therein. In the presence of transcomplementing functions, thedeleted adenovirus vectors of the invention can replicate and packagenew virions.

The term “deleted” as used herein refers to the omission of at least onenucleotide from the indicated region of the adenovirus genome. Deletionscan be greater than about 1, 2, 3, 5, 10, 20, 50, 100, 200, or even 500nucleotides. Deletions in the various regions of the adenovirus genomemay be about at least 1%, 5%, 10%, 25%, 50%, 75%, 90%, 95%, 99%, or moreof the indicated region. Alternately, the entire region of theadenovirus genome is deleted. Preferably, the deletion will prevent oressentially prevent the expression of a functional protein from thatregion. For example, it is preferred that the deletion in the 100Kregion results in the loss of expression of a functional 100K proteinfrom that region In other words, even if there is transcription acrossthe deleted 100K region and translation of the resulting RNAtranscripts, the resulting protein will be essentially non-functional,more preferably, completely non-functional. Alternatively, aninsignificant amount of a function protein is expressed. In general,larger deletions are preferred as these have the additional advantagethat they will increase the carrying capacity of the deleted adenovirusfor a heterologous nucleotide sequence of interest. The various regionsof the adenovirus genome have been mapped and are understood by thoseskilled in the art (see, e.g., FIELDS et al., VIROLOGY, volume 2,chapters 67 and 68 (3d ed., Lippincott-Raven Publishers).

Preferably, the deletions in the inventive adenoviruses are nottemperature-sensitive deletions. In other words, preferably, the loss ofreplication and packaging effected by the deletion(s) are constitutivemutations and are not temperature-sensitive mutations.

As used herein, a “functional” protein is one that retains at least onebiological activity normally associated with that protein. Preferably, a“functional” protein retains all of the activities possessed by thenaturally-occurring protein. A “non-functional” protein is one thatexhibits no detectable biological activity normally associated with theprotein. An “essentially non-functional” protein is one that retainsonly an insignificant amount of biological activity or, alternatively,only an insignificant amount of functional protein is produced.

Adenovirus vectors containing multiple deletions are preferred to bothincrease the carrying capacity of the vector and reduce the likelihoodof recombination to generate replication competent virus. Those skilledin the art will appreciate that where the adenovirus contains multipledeletions, it is not necessary that each of the deletions, if presentalone, would result in a propagation-defective adenovirus as describedabove. As long as one of the deletions renders the adenoviruspropagation-defective, the additional deletions may be included forother purposes, e.g., to increase the carrying capacity of theadenovirus genome for heterologous nucleofide sequences. Preferably,more than one of the deletions prevents the expression of a functionalprotein and renders the adenovirus propagation-defective. Morepreferably, all of the deletions are deletions that would render theadenovirus propagation-defective.

It will further be apparent to those skilled in the art that due to theknown overlap of coding regions within the adenovirus genome, thedeletions described hereinabove may result in the loss of more than onefunctional denovirus protein. Likewise, it is not necessary that allfunctional gene expression from the deleted region be ablated, only thefunctional expression of the 100K, IVa2, polymerase and/or preterminalprotein as indicated. Preferably, the deletions are selected so as notto interfere with other essential functions, e.g., the polymerasedeletions are chosen so as not to interfere with the major late promotersequence or the tripartite leader sequence, and the preterninal proteindeletions are chosen so as not to interfere with the VA-RNA sequences.

It will be apparent that other deletions can be combined with theinventive deletions described herein. For example, first-generationadenovirus vectors are typically deleted for the E1 genes and packagedusing a cell that expresses the E1 proteins (e.g., 293 cells). The E3region is also frequently deleted as well, as there is no need forcomplementation of this deletion. In addition, deletions in the E4, E2a,protein IX, and fiber protein regions have been described, e.g., byArmentano et al, (1997) J. Virology 71:2408, Gao et al., (1 996) J.Virology 70:8934, Dedieu et al., (1 997) J. Virology 71;4626, Wang etal., (1997) Gene Therapy 4:393, U.S. Pat. No. 5,882,877 to Gregory etal. (the disclosures of which are incorporated herein in theirentirety). Preferably, the deletions are selected to avoid toxicity tothe packaging cell. Wang et al., (1997) Gene Therapy 4:393, hasdescribed toxicity from constitutive co-expression of the E4 and E1genes by a packaging cell line. Toxicity may be avoided by regulatingexpression of the E1 and/or E4 gene products by an inducible, ratherthan a constitutive, promoter. Combinations of deletions that avoidtoxicity or other deleterious effects on the host cell can be routinelyselected by those skilled in the art. Those skilled in the art willappreciate that typically, with the exception of the E3 genes, anyadditional deletions will need to be complemented in order to propagate(replicate and package) additional virus, e.g., by transcomplementationwith a packaging cell.

Unlike some other gutted adenovirus vector systems described in theliterature (Clemens et al, (1996) Gene Ther. 3:965; Hardy et al., (1997)J. Virol. 71:1842; Kochanek et al., 1996) Proc. Natl. Acad. Sci. USA93:5731; Kumar-Singh, (1996) Mol. Gen. 5:913; Parks et al., (1996) Proc.Natl. Acad. Sci. USA 93:13565), the vectors of the present invention donot require any type of helper virus for high-titer growth. In fact, useof the multiply-deleted adenoviruses of the present invention inpackaging cells to produce gutted adenovirus vector stocks will reducethe likelihood of any recombination events which produce contaminatingreplication competent virus in gutted adenovirus vector stocks of priorsystems. This is an important problem that may limit the overallusefulness of existing gutted adenovirus preparations (Hardy et al.,(1997) J. Virol. 71:1842). Further, as described in more detail below,use of the deleted adenoviruses of the present invention as helperviruses may increase the efficacy of transduction with gutted adenovirusvectors which may be dependent on the presence of helper viruscontamination for stable gene transduction (Lieber et al., (1997) J.Virol. 70:8944; Lieber et al., (1997) Nature Biotech. 15:1383).

In particular preferred embodiments, the deleted adenoviruses includedeletions in the preterminal protein and the E1 and/or E3 regions. Inother preferred embodiments, the adenoviruses contain deletions in thepolymerase region and the E1 region. Also preferred are adenovirusesincluding deletions in the preterminal protein and polymerase regions aswell as the E1 and/or E3 regions. In addition, adenoviruses carryingdeletions in the 100K and the E1 and/or E3 regions are preferred.Further preferred are deleted adenoviruses containing deletions in thepolymerase and IVa2 as well as the E1 and/or E3 regions. Still morepreferred are adenoviruses containing deletions in the IVa2, polymerase,preterminal protein, and the E1 and/or E3 regions, as well asadenoviruses carrying deletions in the 100K, polymerase, preterminalprotein, and the E1 and/or E3 regions. Yet further preferred areadenoviruses carrying deletions in the IVa2, 100K, polymerase,preterminal protein, and the E1 and/or E3 regions, more preferably still(both the E1 and E3 regions are deleted).

In other particular preferred embodiments, the deletion in the IVa2region is from about nucleotides 4830 to 5766 of the adenovirus serotype5 genome, the deletion in the 100K region is from about nucleotide24,990 to 25,687 of the adenovirus serotype 5 genome, the deletion inthe preterminal protein region is from about nucleotides 9198 to 9630 ofthe adenovirus serotype 5 genome, and the deletion in the polymeraseregion is from about nucleotide 7274 to 7881 of the adenovirus serotype5 genome. Adenoviruses that contain deletions in the preterminal proteinregion at about nucleotides 9198 to 9630 and the polymerase region atabout nucleotide 7274 to 7881 of the adenovirus serotype 5 genome arealso preferred. Further preferred are polymerase and preterminal proteinregion deletions that encompass the region from about nucleotide 7274 to9630 of the adenovirus 5 genome. Those skilled in the art willappreciate that similar deletions can be made in the homologous regionsof the adenovirus genomes from other serotypes. In other particularpreferred embodiments, the deleted adenoviruses are those disclosedherein as [E1-, E3-, pol-]Ad, [E1-, E3-, pol-, TP-]Ad, [E1-, E3-, IVa2-,pol-, pTP-]Ad, [E1-, 100]-Ad, or [E1-, E3-, IVa2-, pol-, pTP-, 100K-]Ad.

As described in more detail hereinbelow, any of the inventiveadenoviruses described above may additionally contain one or moreheterologous nucleotide sequences (e.g., two, three, four, five, six ormore sequences) of interest.

Those skilled in the art will appreciate that the inventive adenovirusvectors may additionally contain other mutations. For example, theadenovirus may be modified or “targeted” as described in Douglas et al.,(1996) Nature Biotechnology 14:1574; U.S. Pat. No. 5,922,315 to Roy etal.; U.S. Pat. No. 5,770,442 to Wickham et al.; and/or U.S. Pat. No.5,712,136 to Wickham et al. (the disclosures of which are allincorporated herein in their entirety).

II. Reagents and Methods for Producing Deleted Adenovirus.

Except as otherwise indicated, standard methods may be used for theconstruction of the recombinant adenovirus genomes, helper adenoviruses,and packaging cells according to the present invention. Such techniquesare known to those skilled in the art. See, e.g., SAMBROOK et al.,MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor,N.Y., 1989); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(Green Publishing Associates, Inc. and John Wiley & Sons, Inc., NewYork).

The inventive deleted adenovirus vectors can be generated as describedherein or by any other method known in the art. For example, deletedadenoviruses can be generated by co-transfection of a shuttle plasmidcontaining a deletion(s) of interest (and optionally a heterologousnucleotide sequence) and either a plasmid encoding the remainingsequences of the adenovirus or with virion DNA from a viable adenovirusinto an appropriate packaging cell. Co-transfection of the two moleculesinto the packaging cell followed by a successful recombination eventbetween the two molecules (the shuttle plasmid also contains regions ofhomology to the adenovirus genome) results in the generation of the fulllength vector genome, containing the deletions of interest and capableof propagation in the appropriate transcomplementing cell.

According to one particular method a plasmid is created containing asubstantial portion of the adenovirus genome which includes the desirednew deletion(s) as well as other desired mutations (preferably alsodeletions). The starting plasmid preferably contains, in addition to mu0 through 1 of the adenovirus genome, a restriction enzyme site (e.g.,an Ascl site), and mu 9 through at least mu 40 of the adenovirus genome.Such a starting plasmid may be manipulated using techniques known in theart to contain the desired additional deletions and/or the desiredtransgene.

The resulting plasmid containing the desired mutations in the adenovirusgenome portion of the plasmid and/or the desired transgene is thenlinearized by digesting with a restriction endonuclease, such as EcoRI,BspHI, NheI, or AseI. The linearized plasmid DNA containing the desiredtransgene and/or deletion is cotransfected with adenoviral DNA, whichhas been prepared as described below, into a packaging cell line capableof supporting (transcomplementing) a productive infection of adenoviruswith the desired deletion(s).

The adenovirus genomic DNA used in cotransfection can be isolated asintact virion DNA-terminal protein complex as described in Jones et al.,(1978) Cell 13:181. The genomic DNA may be from a virus which alreadycontains desired mutations, preferably deletions, such as in the E1, E3,pol, pTP, IVa2, and/or 100K regions. Prior to its use in cotransfection,adenovirus genomic DNA is first digested with at least two restrictionenzymes. These enzymes are selected to cause a significant number ofdouble stranded breaks in the adenoviral genome in the area of thechromosome for which no recombination events are desired. Preferably,the area where no recombination events are desired includes at leastnucleotide 1 through at nucleotide 10590, allowing recombination eventsto occur, for example, between nucleotide 10590 and nucleotide 15671 inpAdAscL-Δpol (Example 2).

Use of a shuttle plasmid containing a larger portion of the adenovirusgenome such as that present in one of the preferred shuttles of thepresent invention, pAdAscL-Δpol (Example 2), promotes recombination inthis more distal region of the adenoviral genome. Further, by firstdigesting the genomic adenoviral DNA using multiple restriction enzymes,preferably Clal, Xbal, and Scal, one achieves a significant reduction inthe ability of the genomic adenoviral DNA to generate a viable,non-recombinant virus. This is due to the fact that the portion of theviral DNA required for such recombination is more likely to berestriction enzyme digested multiple times. The utilization of severalrestriction enzymes therefore essentially prevents the generation ofundesirable, non-recombinant virus derived from the digested virion DNA.

The preferred shuttling plasmid is pAdAscL-Δpol (Example 2). Thisplasmid is similar to pAdAscl, a plasmid known to the prior art, howeverpAdAsci contains only map units 9 through 16 of the adenovirus genomewhile pAdAscL-wt contains map units 9 through 43. The additional Adsequences present in pAdAscL-wt allows one to digest virion DNA withmultiple restriction enzymes prior to cotransfection with the linearizedshuttle plasmid during initial vector construction, thereby reducingcreation by recombination of undesirable recombinant viruses.

The genomic DNA can preferably be isolated from an adenovirus strainwhich contains at least the E1 deletion as well as, optionally, otherdesired, previously created deletions to further reduce the possibilityof generating adenovirus without the desired genetic attributes.

Thus, the manipulated plasmid DNA, containing the desired transgeneand/or mutation, is linearized (i.e. with BspHI, EcoRI, or NheI),co-transfected with the multiply-digested virus genome, preferably theAd5 adenovirus E1-strain dl7001 as described in Example 1 below, into apackaging cell line which expresses any required adenovirus genefunctions. After incubating for a time sufficient to allow for viralgrowth, the transfected cells are harvested and mixed withnon-transfected packaging cells. This cell mixture is distributed totissue culture cluster plates and, after incubation, the individualwells of these plates which demonstrate viral cytopathic effect (CPE)are harvested. Each well represents a single clonal isolate of virus.The clonal isolates are then characterized as described elsewhereherein. For example, DNA restriction mapping, functional studies, ³²Pprobes and similar studies are performed to confirm and characterize thegenotype of the recombinant vector/virus.

The present invention further provides reagents (e.g., isolatednucleotide sequences, vectors, cells) and methods for producing theinventive deleted adenoviruses.

Further disclosed herein are isolated nucleotide sequences, morepreferably DNA sequences, encoding a deleted adenovirus 100K protein.The nucleotide sequence encoding the 100K protein contains one or moredeletions (as described for deleted adenovirus vectors hereinabove) suchthat the deletions(s) essentially prevents the expression of afunctional 100K protein from the deleted nucleotide sequence (asdescribed hereinabove). Preferably, the nucleotide sequence is containedwithin an adenovirus genome, more preferably a recombinant adenovirusgenome. Also provided by the present invention are vectors (e.g., aplasmid vector) carrying the adenovirus genome containing the deleted100K region. An illustrative plasmid according to the present inventionis the plasmid disclosed herein as pcDNA3+100K. Further provided arecells containing the inventive vectors, including bacterial, protozoan,yeast, fungus, plant and animal (e.g., insect, avian and mammalian)cells.

Also provided are isolated nucleotide sequences (preferably, DNAsequences), adenovirus genomes, and vectors encoding an adenovirus IVa2protein containing one or more deletions (as described hereinabove fordeleted adenovirus vectors) and cells containing the same. Thesereagents are otherwise the same as those described for the 100K proteinin the preceding section. Exemplary plasmids encoding a deleted IVa2gene include those described herein as pAdAscLΔIVa2 (FIG. 19; deletionof about 942 bp at about nucleotides 4830 to 5766 of the Ad5 genome inaddition to deleted E1 and E3 regions), pAdAscLΔIVa2, Δpol (FIG. 38;deletion of about 942 bp at about nucleotides 4830 to 5766 and adeletion of about 608 bp at about nucleotides 7274 to 7881 of the Ad5genome in addition to deleted E1 and E3 regions), pAdAscLΔIVa2, Δpp(1.6) (FIG. 36; eletion of about 942 bp at about nucleotides 4830 to5766, a deletion of bout 608 bp at about nucleotides 7274 to 7881, and adeletion of about 955 bp at about nucleotides 8631 to 9585 of the Ad5genome in addition to deleted E1 and E3 regions), and pAdAscLΔIV2, Δpp(2.4) (FIG. 37; deletion of about 942 bp at about nucleotides 4830 to5766, a deletion of about 608 bp at about nucleotides 7274 to 7881, anda deletion of about 2312 bp at about nucleotides 7274 to 9585 of the Ad5genome in addition to deleted E1 and E3 regions).

The present invention further encompasses cells for packaging theinventive deleted adenoviruses. Accordingly, in one particularembodiment, the present invention provides a cell containing an isolatednucleotide sequence encoding a functional (as described hereinabove)adenovirus 100K protein. The cell may be a bacterial, protozoan, yeast,fungus, plant or animal cell.

Preferably, the cell is an animal cell (e.g. an insect, avian ormammalian cell), more preferably a mammalian cell. The nucleotidesequence can be present in the cell extrachromosomally or, preferably,may be stably integrated into the genome of the cell. Typically, theisolated nucleotide sequence encoding the functional 100K protein isoperatively associated with appropriate expression control sequences(e.g., a promoter sequence). Expression of the nucleotide sequence canbe constitutive or inducible.

In particular preferred embodiments, the cell expressing the functional100K protein can transcomplement a 100K deleted adenovirus genome (asdescribed hereinabove). In other words, a propagation-defective 100Kdeleted adenovirus can be replicated and packaged in the cell expressingthe functional 100K protein. in preferred embodiments, the cell is aK-16 cell or a C7 cell constitutively expressing the adenovirus 100Kprotein, each as disclosed herein.

Those skilled in the art will appreciate that the deleted adenovirusgenome may contain additional deletions in addition to the 100Kdeletion. Typically, with the exception of the E3 region, it will benecessary to transcomplement these deletions in order to package newvirus particles. Transcomplementation can be achieved by any means knownin the art, e.g., by the packaging cell or with a helper adenovirus. Asa further alternative, the missing function may be transientlytransfected into the packaging cell that normally does not express thisfunction. Preferably, the inventive adenoviruses are produced with atranscomplementing packaging cell.

In another particular embodiment, the present invention provides a cellcontaining an isolated nucleotide sequence encoding a functional (i.e.,biologically active) adenovirus IVa2 protein. Cells containing theisolated nucleotide sequence encoding the functional IVa2 protein are asdescribed above for cells expressing a functional 100K protein.Exemplary cells expressing a functional IVa2 protein include thosedisclosed herein as a B6 or C7 cell.

The present invention also encompasses methods of producing the deletedadenovirus particles of the present invention. According to oneparticular method a deleted adenovirus is introduced into atranscomplementing packaging cell (as described above). The adenovirusgenome contains one or more deletions in one or more of the adenovirus100K, IVa2 and/or preterminal protein regions (as described in detailfor deleted adenovirus vectors hereinabove). Methods of transducingcells with adenoviruses are well-known by those skilled in the art. Thedeleted adenovirus is replicated and packaged in the transcomplementingcell (i.e., expressing the deleted function(s)), and the deletedadenovirus particles are collected. The packaging cell is preferably ananimal cell (e.g., insect, avian, mammalian), more preferably, amammalian cell.

In an alternate embodiment, the present invention provides a method ofproducing a deleted adenovirus that contains one or more deletions inthe E1 region and one or more deletions in the polymerase region (asdescribed hereinabove for deleted adenoviruses). The deleted adenoviruscan replicated and packaged in a transcomplementing cell expressing thedeleted adenovirus functions the new virions collected.

In another preferred embodiment, the present invention provides a methodfor producing a deleted adenovirus containing two or more deletions intwo or more regions of the adenovirus genome, wherein one of the deletedregions is in the polymerase region. In particular embodiments whereinthere are two deleted regions, the first deleted region is thepolymerase region and the second deleted region is any other region asdescribed herein, with the exception of the E3 region. In more preferredembodiments, the adenovirus has one or more deletions in the polymeraseregion and one or more deletions in the E1 region, and optionally, oneor more deletions in other regions of the adenovirus genome. The deletedadenovirus can replicated and packaged in a transcomplementing cellexpressing the deleted adenovirus functions the new virions collected.

As a further non-limiting alternative, the present invention provides amethod of producing a deleted adenovirus carrying a nucleotide sequenceencoding a GAA protein and further containing one or more deletions inone or more of the 100K, IVa2, preterminal protein, and polymeraseregions. The deleted adenovirus can replicated and packaged in atranscomplementing cell expressing the deleted adenovirus functions thenew virions collected.

PCT Publication WO 98/17783 to Chamberlain et al. describes packaging ofadenovirus vectors containing deletions in the polymerase and/orpreterminal protein regions. However, as described herein and byAmalfitano et al., (1998) J. Virology 72:926 (page 928, first fullparagraph of Methods & Materials), the methods described in thisreference based on the pBHG11 plasmid failed to produce adenovirusesdeleted in the polymerase and/or preterminal protein regions.

In contrast, according to the inventive packaging methods describedherein, the collected adenovirus preferably has a titer of at least 100infectious units per cell, more preferably at least 1000 infectiousunits per cell, more preferably still at least 10,000 infectious unitsper cell. The present inventors have successfully used the inventivemethods described herein to produce high titer stocks of adenovirusescarrying deletions in the E1, E3, 100K, IVa2, preterminal protein and/orpolymerase regions.

The present invention further encompasses methods of producing theinventive adenovirus vectors using bacterial cells. For example, He etal. (1998) Proc. Natl. Acad Sci. USA 95:2509 describe a method forproducing deleted adenovirus using an adenoviral plasmid (see also, U.S.Pat. No. 5,922,576 to He et al., the disclosure of which is incorporatedherein in its entirety). Accordingly, in one particular embodiment, thepresent invention provides a method of producing an infectious deletedadenovirus of the present invention by introducing a bacterial plasmidcarrying an adenovirus genome into a bacterial cell. The adenovirusgenome comprises one or more deletions in one or more of the polymerase,preterminal protein, 100K, and/or IVa2 regions (as described hereinabovefor deleted adenoviruses). The bacterial plasmid is amplified in thebacterial cell, recovered, and linearized. The linearized plasmidcarrying the deleted adenovirus genome is introduced into atranscomplementing packaging cell (as described hereinabove) where it isreplicated and packaged into new virions. The adenovirus particles arecollected. Preferably, the adenovirus genome further encodes one or moreheterologous nucleotide sequences.

The present invention further provides a method for producing a guttedadenovirus vector containing an adenovirus minichromosome. Adenovirusminichromosomes are as described by WO 98/17783 to Chamberlain et al.and Kumar-Singh et al., (1996) Human Molecular Genetics 5:913. Thedeleted adenoviruses of the present invention can be used as anoptimized helper virus for the growth of gutted adenoviruses. A methodfor growing high titer stocks of a gutted adenovirus vectors using thedeleted viruses of the present invention involves co-infecting guttedadenovirus vectors and a propagation-defective deleted adenovirus of thepresent invention into a packaging cell that is permissive for thegrowth of the deleted vector, then harvesting the resulting virusparticles.

According to one particular and preferred method, a plasmid containingat least one adenovirus ITR (preferably two ITRs), an adenoviruspackaging sequence, and one or more heterologous nucleotide sequences ofinterest are introduced into a packaging cell. A helper adenoviruscomprising an adenovirus genome containing one or more deletions in the100K region is also introduced into the packaging cell. The 100K deletedvector may also contain additional deletions in other regions, asdescribed above. The packaging cell expresses a functional 100K proteinand transcomplements the deletion in the 100K region of the adenovirusgenome. The packaging cell and 100K deleted adenovirus genome are eachas described hereinabove. The adenovirus minichromosome can bereplicated and packaged in the presence of the helper adenovirus andtranscomplementing cell line, and the gutted adenovirus vectorscontaining the adenovirus minichromosome are collected.

If desired, the gutted vector may be separated from the deletedadenovirus by any method known in the art, e.g., cesium chloridecentrifugation or any other density gradient. In addition to use ofprior art separation techniques, one can reduce the helper viruscontamination by preventing packaging of the helper virus. For example,the adenovirus genome carried by the helper adenovirus may lack anadenovirus packaging sequence to prevent packaging of the helperadenovirus genome. Alternatively, the helper adenovirus carries amodified packaging sequence that does not enable encapsidation of thehelper adenovirus genome.

As a further non-limiting alternative, the helper adenovirus genome haslox sites flanking the packaging sequence and the packaging cellproduces the Cre recombinase protein. The presence of the Crerecombinase results in lox mediated recombination and removal of allsequences flanked by the lox sites (e.g., the packaging signal and/orother adenovirus sequences) and prevents the lox-containing DNA frombeing packaged (Parks et al., (1996) Proc. Natl. Acad. Sci. USA93:13565). In a variation of this methodology the lox sites are placedwithin the multiply deleted vector flanking a greater amount of the Adgenome. When placed into Cre cells a large portion of the deleted vectorgenome is removed. The resulting reduced helper virus is purified awayfrom the unloxed vector via cesium chloride centrifugation or othermethods known in the art (Lieber et al., (1996) J. Virol. 70:8944;Kochanek et al., (1996) Proc. Natl. Acad. Sci. USA 93:5731).

Also provided herein are methods of producing a gutted adenoviruscarrying a minichromosome with a helper adenovirus containing anadenovirus genome comprising one or more deletions in the IVa2 regionand a packaging cell that transcomplements this deletion by expressing afunctional IVa2 protein. These methods are otherwise similar to thosedescribed above for packaging a gutted adenovirus using a helperadenovirus containing a deleted 100K region.

Further provided herein are methods of producing a gutted adenoviruscarrying a minichromosome with a helper adenovirus containing anadenovirus genome comprising one or more deletions in the polymeraseand/or preterminal protein regions and a packaging cell thattranscomplements this deletion(s) by expressing a functional preterminalprotein and/or polymerase, respectively. Preterminal protein andpolymerase deletions are as described above for deleted adenovirusvectors. These methods are otherwise similar to those described abovefor packaging a gutted adenovirus using a helper adenovirus containing adeleted 100K region.

The deleted adenoviruses of the present invention can also be used ingene therapy methods using gutted adenovirus vectors to assist instabilizing the gutted vector genomes in the target cells byco-infecting the target cells with deleted viruses of the presentinvention along with the gutted vector. Destabilization of guttedadenovirus vectors due to lack of helper virus contamination (andtrans-acting activities) has recently been described as a potentiallysevere limitation of gutted Ad vector preparations (Lieber et al.,(1996) J. Virol. 70:8944). Contamination of the gutted adenovirus stockwith the deleted vectors described herein is preferable tofirst-generation E1 deleted helpers, which have been reported to be“leaky” when introduced into cells or subjects. Preferably, according tothis embodiment, the contaminating helper adenovirus is amultiply-eleted adenovirus, so as to minimize the likelihood ofrecombination to generate replication competent vector and/or any“leakiness” in gene expression.

Accordingly, the present invention provides a composition (preferably, apharmaceutical composition) comprising a gutted adenovirus containing aminichromosome and a deleted helper adenovirus according to the presentinvention. The abundance of the deleted helper adenovirus is preferablyless than about 50%, 30%, 20%, 10%, 5%, 2-5%, 1%, 0.05%, 0.01%, 0.001%or lower than the abundance of the gutted adenovirus in the composition.These compositions can be advantageously administered to a subjectaccording to any of the methods described herein to combine theadvantages of a gutted adenovirus vector with the stabilizing effect ofthe deleted helper adenovirus.

III. Recombinant Adenovirus Vectors.

As used herein, a “recombinant adenovirus vector” is an adenovirusvector that carries one or more heterologous nucleotide sequences ortransgenes (e.g., two, three, four, five or more heterologous nucleotidesequences or transgenes). The deleted adenovirus vectors of the presentinvention are useful for the delivery of nucleic acids to cells both invitro and in vivo. In particular, the inventive vectors can beadvantageously employed to deliver or transfer nucleic acids to animal,more preferably mammalian, cells. Nucleic acids of interest includenucleic acids encoding peptides and proteins, preferably therapeutic(e.g, for medical or veterinary uses) or immunogenic (e.g., forvaccines) peptides or proteins.

Alternatively, in particular embodiments of the invention, the nucleicacid of interest may encode an antisense nucleic acid, a ribozyme, orother non-translated RNAs, such as “guide” RNAs (Gorman et al., (1998)Proc. Nat. Acad. Sci. USA 95:4929), and the like.

The present invention also provides vectors useful as vaccines. Theantigen can be presented in the adenovirus capsid, alternatively, theantigen can be expressed from a heterologous nucleic acid introducedinto a recombinant adenovirus genome and carried by the inventiveadenoviruses. Any immunogen of interest can be provided by theadenovirus vector. Immunogens of interest are well-known in the art andinclude, but are not limited to, immunogens from human immunodeficiencyvirus (e.g., envelope proteins), influenza virus, gag proteins, cancerantigens, HBV surface antigen (to immunize against hepatitis), rabiesglycoproteins, and the like.

As a further alternative, the adenovirus vectors can be used to infect acell in culture to express a desired gene product, e.g., to produce aprotein or peptide of interest (for example, lysosomal acidα-glucosidase). Preferably, the protein or peptide is secreted into themedium and can be purified therefrom using routine techniques known inthe art. Signal peptide sequences that direct extracellular secretion ofproteins are known in the art and nucleotide sequences encoding the samecan be operably linked to the nucleotide sequence encoding the peptideor protein of interest by routine techniques known in the art.Alternatively, the cells can be lysed and the expressed recombinantprotein can be purified from the cell lysate. The cell may be abacterial, protozoan, plant, yeast, fungus, or animal cell. Preferably,the cell is an animal cell (e.g., insect, avian or mammalian), morepreferably a mammalian cell. Also preferred are cells that are competentfor transduction by adenoviruses.

The size of the adenovirus vector can vary. Generally, the adenovirusgenome is most stable at sizes of about 28 kb to 38 kb. (approximately75% to 105% of the native genome size). In the case of an adenovirusvector containing large deletions and a relatively small transgene,“stuffer DNA” can be used to maintain the total size of the vectorwithin the desired range by methods known in the art. The deletedvectors of the present invention can advantageously be used to delivertransgenes of up to about 9, 11 or even 12.5 kb in size or more.

Those skilled in the art will appreciate that the heterologousnucleotide sequence(s) are preferably operably associated with theappropriate expression control sequences. For example, the recombinantadenovirus vectors of the invention preferably contain appropriatetranscriptionttranslation control signals and polyadenylation signalsoperably associated with the heterologous nucleic acid sequencers) to bedelivered to the target cell. Those skilled in the art will appreciatethat a variety of promoter/enhancer elements may be used depending onthe level and tissue-specific expression desired. The promoter can beconstitutive or inducible (e.g., the metallothionine promoter),depending on the pattern of expression desired. The promoter may benative or foreign and can be a natural or a synthetic sequence. Byforeign, it is intended that the transcriptional initiation region isnot found in the wild-type host into which the transcriptionalinitiation region is introduced. The promoter is chosen so that it willfunction in the target cell(s) of interest. Brain-specific,hepatic-specific, and muscle-specific (including skeletal, cardiac,smooth, and/or diaphragm-specific) promoters are more preferred.Mammalian promoters are also preferred.

More preferably, the heterologous nucleotide sequencers) are operativelyassociated with a cytomegalovirus (CMV) major immediate-early promoter,an albumin promoter, an Elongation Factor 1-α (EF1-α) promoter, a PγKpromoter, a MFG promoter, or a Rous sarcoma virus promoter. It has beenspeculated that driving heterologous nucleotide transcription with theCMV promoter results in down-regulation of expression in immunocompetentanimals (see, e.g., Guo et al., (1996) Gene Therapy 3:802). Accordingly,it is also preferred to operably associate the heterologous nucleotidesequence(s) with a modified CMV promoter that does not result in thisdown-regulation of transgene expression.

In embodiments wherein there is more than one heterologous nucleotidesequence, those skilled in the art will appreciate that the heterologousnucleotide sequences may be operatively associated with a singleupstream promoter and one or more downstream internal ribosome entrysite (IRES) sequences (e.g., the picornavirus EMC IRES sequence).

Also preferred are embodiments wherein the adenovirus genome contains atleast one adenovirus inverted terminal repeat (ITR) sequence, morepreferably two adenovirus ITR sequences. Moreover, it is also preferredthat the recombinant adenovirus genome carrying the transgene alsoencodes an adenovirus packaging sequence. For example, in one particularembodiment, the adenovirus genome comprises one or more heterologousnucleotide sequences, the 5′ and 3′ adenovirus ITRs, the adenoviruspackaging sequence, and an adenovirus E1A enhancer sequence.

In embodiments of the invention in which the heterologous nucleotidesequence(s) will be transcribed and then translated in the target cells,specific initiation signals are generally required for efficienttranslation of inserted protein coding sequences. These exogenoustranslational control sequences, which may include the ATG initiationcodon and adjacent sequences, can be of a variety of origins, bothnatural and synthetic.

Heterologous nucleotide sequences encoding proteins and peptides includethose encoding reporter proteins (e.g., an enzyme). Reporter proteinsare known in the art and include, but are not limited to, GreenFluorescent Protein, β-galactosidase, alkaline phosphatase, andchloramphenicol acetyltransferase gene. More preferably, the adenovirusvector is the vector disclosed herein as AdLacZΔpol or AdLacZΔpp.

Therapeutic peptides and proteins include, but are not limited to,cystic fibrosis transmembrane regulator protein (CFTR), dystrophin(including the protein product of dystrophin mini-genes, see, e.g,Vincent et al., (1993) Nature Genetics 5:130), utrophin (Tinsley et al.,(1996) Nature 384:349), clotting factors (e.g., Factor XIII, Factor IX,Factor X, etc.), erythropoietin, the LDL receptor, lipoprotein lipase,ornithine transcarbamylase, β-globin, α-globin, spectrin, α-antitrypsin,adenosine deaminase, hypoxanthine guanine phosphoribosyl transferase,β-glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase,branched-chain keto acid dehydrogenase, hormones, growth factors,cytokines (e.g., interferon-γ, interleukin-2, interleukin-4,granulocyte-macrophage colony stimulating factor), suicide gene products(e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumornecrosis factor), proteins conferring resistance to a drug used incancer therapy, tumor suppressor gene products (e.g., p53, Rb, Wt-1),and any other peptide or protein that has a therapeutic effect in asubject in need thereof.

In particular preferred embodiments of the invention, the heterologousnucleotide sequence encodes a protein or peptide that is associated witha metabolic disorder. By “associated with a metabolic disorder”, it isintended that the expressed protein or peptide is one that is deficientor defective in a metabolic disorder, or is otherwise a causative agentin a metabolic disorder.

In other particular preferred embodiments, the protein or peptide is alysosomal protein or peptide, more preferably a precursor protein orpeptide that retains the mannose-6-phosphate residues that arecharacteristic of proteins targeted to the lysosomal compartment.

In still further preferred embodiments, the heterologous nucleotidesequence encodes a peptide or protein that is associated with alysosomal storage disease. By “associated with a lysosomal storagedisease”, it is intended that the expressed protein or peptide is onethat is deficient or defective in a lysosomal storage disorder, or isotherwise a causative agent in a lysosomal storage disorder.

There are a multitude of lysosomal storage diseases, as is well-known inthe art. Exemplary lysosomal storage disease include, but are notlimited to, GM1 gangliosidosis, Tay-Sachs disease, GM2 gangliosidosis(AB variant), Sandhoff disease, Fabry disease, Gaucher disease,metachromatic leukodystrophy, Krabbe disease, Niemann-Pick disease(Types A-D), Farber disease, Wolman disease, Hurler Syndrome (MPS III),Scheie Syndrome (MPS IS), Hurler-Scheie Syndrome (MPS IH/S), HunterSyndrome (MPS II), Sanfilippo A Syndrome (MPS IIIA), Sanfilippo BSyndrome (MPS IIIB), Sanfilippo C Syndrome (MPS IIIC), Sanfilippo DSyndrome (MPS IIID), Morquio A disease (MPS IVA), Morquio B disease (MPSIVB), Maroteaux-lmay disease (MPS VI), Sly Syndrome (MPS VII),α-mannosidosis, β-mannosidosis, fucosidosis, aspartylglucosaminuria,sialidosis (mucolipidosis I), galactosialidosis (Goldberg Syndrome),Schindler disease, mucolipidosis II (I-Cell disease), mucolipidosis III(pseudo-Hurler polydystrophy), cystinosis, Salla disease, infantilesialic acid storage disease, Batten disease (juvenile neuronal ceroidlipofuscinosis), infantile neuronal ceroid lipofuscinosis, mucolipidosisIV, and prosaposin.

Proteins or peptides that are associated with lysosomal storage diseasesaccording to the present invention include, but are not limited to,β-galactosidase, β-hexosaminidase A, β-hexosaminidase B, GM₂ activatorprotein, glucocerebrosidase, arylsulfatase A, galactosylceramidase, acidsphingomyelinase, acid ceramidase, acid lipase, α-L-iduronidase,iduronate sulfatase, heparan N-sulfatase, α-N-acetylglucosaminidaseacetyl-CoA, glucosaminide acetyltransferase,N-acetylglucosamine-6-sulfatase, arylsulfatase B, β-glucuronidase,α-mannosidase, β-mannosidase, β-L-fucosidase,N-aspartyl-β-glucosaminidase, α-neuraminidase, lysosomal protectiveprotein, α-N-acetyl-galactosaminidase,N-acetylglucosamine-1-phosphotransferase, cystine transport protein,sialic acid transport protein, the CLN3 gene product, palmitoyl-proteinthioesterase, saposin A, saposin B, saposin C, and saposin D.

The present invention further provides deleted recombinant adenovirusvectors carrying a transgene encoding a protein or peptide associatedwith a glycogen storage disease. By “associated with a glycogen storagedisease”, it is intended that the expressed protein or peptide is onethat is deficient or defective in a glycogen storage disease, or isotherwise a causative agent in a lysosomal storage disease.

There are a multitude of glycogen storage diseases (GSD), as iswell-known in the art. Exemplary glycogen storage diseases include, butare not limited to, Type Ia GSD (von Gierke disease), Type Ib GSD, TypeIc GSD, Type Id GSD, Type II GSD (including Pompe disease or infantileType II GSD), Type IIIa GSD, Type IIIb GSD, Type IV GSD, Type V GSD(McArdle disease), Type VI GSD, Type VII GSD, glycogen synthasedeficiency, hepatic glycogenosis with renal Fanconi syndrome,phosphoglucoisomerase deficiency, muscle phosphoglycerate kinasedeficiency, phosphoglycerate mutase deficiency, and lactatedehydrogenase deficiency.

Proteins or peptides that are associated with glycogen storage diseasesaccording to the present invention include, but are not limited to,glucose 6-phosphatase, lysosomal acid a glucosidase, glycogendebranching enzyme, branching enzyme, muscle phosphorylase, liverphosphorylase, phosphorylase kinase, muscle phosphofructokinase,glycogen synthase, phosphoglucoisomerase, muscle phosphoglyceratekinase, phosphoglycerate mutase, and lactate dehydrogenase.

In more preferred embodiments, the deleted recombinant adenovirus vectorcarries a transgene encoding a lysosomal acid α-glucosidase (GAA), e.g.,to treat Type II GSD including infantile (Pompe disease), juvenile andadult onset forms of the disease. More preferably, the lysosomal acidα-glucosidase is a human lysosomal acid α-glucosidase (hGAA). Thetransgene may encode either the mature GAA protein (e.g., the 76 kDform) or a GAA precursor (e.g., the 110 kD form). Preferably, thetransgene encodes a GAA precursor. The term “GAA” as used hereinencompasses mature and precursor GAA proteins as well as modified (e.g.,truncated or mutated) GAA proteins that retain biological function(i.e., have at least one biological activity of the native GAA protein,e.g., can hydrolyze glycogen).

Lysosomal acid α-glucosidase (E.C. 3.2.1.20) (1,4-α-D-glucanglucohydrolase), is an exo-1,4α-D-glucosidase that hydrolyses both α-1,4and α-1,6 linkages of oligosaccharides liberating glucose. It catalyzesthe complete degradation of glycogen with slowing at branching points.The 28 kb acid α-glucosidase gene on chromosome 17 encodes a 3.6 kb mRNAwhich produces a 951 amino acid polypeptide (Hoefsloot et al., (1988)EMBO J. 7:1697; Martiniuk et al., (1990) DNA and Cell Biology 9:85). Thenucleotide sequence of a cDNA coding for the polypeptide, as well as thededuced amino acid sequence is provided in Hoefsloot et al. (Id.). Thefirst 27 amino acids of the polypeptide are typical of a leader sequenceof a signal peptide of lysosomal and secretory proteins. The enzymereceives co-translational N-linked glycosylation on the endoplasmicreticulum. It is synthesized as a 110-kDa precursor form, which maturesby extensive modification of its glycosylation, and phosphorylation andby proteolytic processing through an approximately 90-kDa endosomalintermediate into the final lysosomal 76 and 67 kDa forms (Hoefsloot,(1988) EMBO J. 7:1697; Hoefsloot et al., (1990) Biochem. J. 272:485;Wisselaar et al., (1993) J. Biol. Chem. 268:2223; Hermans et al., (1993)Biochem. J. 289:681).

The human GAA gene as described by Hoefsloot et al., (1988) EMBO J.7:1697 and Van Hove et al., (1996) Proc. Natl. Acad. Sci. USA 93:65,includes 5′ untranslated sequences. In particular preferred embodiments,the hGAA transgene includes the entire approximately 3.8 kb sequencedescribed by Van Hove et al. Alternatively, the deleted adenoviruses ofthe present invention may encode more or less of the 5′ and 3′untranslated regions of the GAA gene. In other preferred embodiments,the heterologous nucleotide sequence is the approximately 3.3 kbnucleotide sequence encoding the full-length GAA precursor but lackingessentially all of the 5′ and 3′ sequences (see, e.g., Example 23, theh5′sGAA sequence) or the 3.8 kb GAA sequence from pcDNA-GAA (Example 23)containing additional 5′ untranslated sequences.

Also preferred are the adenovirus vectors disclosed herein as AdhGAAΔpol(3.8 kb), Ad/EF1-α/hGAAΔpol (3.8 kb), AdhGAAΔpp (3.8 kb),Ad/EF1-α/hGAAΔpp (3.8 kb), Adh5′sGAAΔpol (3.3 kb), Ad/EF1-α/h5′sGAAΔpol(3.3 kb), AdhGAAΔpp (3.3 kb), and Ad/EF1-α/h5′sGAAΔpp (3.3 kb).

IV. Gene Transfer Technology.

The methods of the present invention provide a means for deliveringheterologous nucleotide sequences into a broad range of host cells,including both dividing and non-dividing cells in vitro or in vivo. Thevectors, methods and pharmaceutical formulations of the presentinvention are additionally useful in a method of administering a proteinor peptide to a subject in need thereof, as a method of treatment orotherwise. In this manner, the protein or peptide may thus be producedin vivo in the subject. The subject may be in need of the protein orpeptide because the subject has a deficiency of the protein or peptide,or because the production of the protein or peptide in the subject mayimpart some therapeutic effect, as a method of treatment or otherwise,and as explained further below.

Gene transfer has substantial potential use in understanding andproviding therapy for disease states. There are a number of inheriteddiseases in which defective genes are known and have been cloned. Insome cases, the function of these cloned genes is known. In general, theabove disease states fall into two classes: deficiency states, usuallyof enzymes, which are generally inherited in a recessive manner, andunbalanced states, at least sometimes involving regulatory or structuralproteins, which are inherited in a dominant manner. For deficiency statediseases, gene transfer could be used to bring a normal gene intoaffected tissues for replacement therapy, as well as to create animalmodels for the disease using antisense mutations. For unbalanced diseasestates, gene transfer could be used to create a disease state in a modelsystem, which could then be used in efforts to counteract the diseasestate. Thus the methods of the present invention permit the treatment ofgenetic diseases. As used herein, a disease state is treated bypartially or wholly remedying the deficiency or imbalance that causesthe disease or makes it more severe. The use of site-specificintegration of nucleic sequences to cause mutations or to correctdefects is also possible.

In general, the present invention can be employed to deliver any foreignnucleotide sequence to treat or ameliorate the symptoms associated withany disorder related to gene expression. Illustrative disease statesinclude: lysosomal storage diseases, glycogen storage diseases,hemophilias (e.g., hemophilia A and hemophilia B) and other clottingdisorders, Gaucher's Disease, diabetes mellitus, cystic fibrosis (andother diseases of the lung), muscular dystrophies (e.g., Duchenne,Becker), diseases of the nervous system (e.g., Alzheimer's Disease,Parkinson's Disease, amyotrophic lateral sclerosis, epilepsy), retinaldegenerative diseases (and other diseases of the eye), diseases of solidorgans (e.g., brain, liver, kidney, heart), and any other diseaseshaving an infectious or genetic basis.

The instant invention can also be employed to provide an antisensenucleic acid to a cell in vitro or in vivo. Expression of the antisensenucleic acid in the target cell diminishes expression of a particularprotein by the cell. Accordingly, antisense nucleic acids can beadministered to decrease expression of a particular protein in a subjectin need thereof. Antisense nucleic acids can also be administered tocells in vitro to regulate cell physiology, e.g., to optimize cell ortissue culture systems. The present invention is also useful to deliverother non-translated RNAs, e.g., ribozymes or “guide” RNAs (see, e.g.,Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929) to a targetcell.

Finally, the instant invention finds further use in diagnostic andscreening methods, whereby a gene of interest is transiently or stablyexpressed in a cell culture system.

V. Subjects, Pharmaceutical Formulations, Vaccine and Modes ofAdministration.

The present invention finds use in veterinary and medical applications.Suitable subjects include both avians and mammals, with mammals beingpreferred- The term “avian” as used herein includes, but is not limitedto, chickens, ducks, geese, quail, turkeys and pheasants. The term“mammal” as used herein includes, but is not limited to, humans,bovines, ovines, caprines, equines, felines, canines, lagomorphs, etc.Human subjects are the most preferred. Human subjects include neonates,infants, juveniles, and adults.

In particular embodiments, the present invention provides apharmaceutical composition comprising a virus particle of the inventionin a pharmaceutically-acceptable carrier or other medicinal agents,pharmaceutical agents, carriers, adjuvants, diluents, etc. Forinjection, the carrier will typically be a liquid. For other methods ofadministration, the carrier may be either solid or liquid, such assterile, pyrogen-free water or sterile pyrogen-free phosphate-bufferedsaline solution. For inhalation administration, the carrier will berespirable, and will preferably be in solid or liquid particulate form.As an injection medium, it is preferred to use water that contains theadditives usual for injection solutions, such as stabilizing agents,salts or saline, andlor buffers.

By “pharmaceutically acceptable” it is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject along with the viral vector without causingany undesirable biological effects. Thus, such a pharmaceuticalcomposition can be used, for example, in transfection of a cell ex vivoor in administering a viral particle directly to a subject.

Vaccines of the present invention comprise an immunogenic amount ofinfectious virus particles as disclosed herein in combination with apharmaceutically-acceptable carrier. An “immunogenic amount” is anamount of the infectious virus particles that is sufficient to evoke animmune response in the subject to which the pharmaceutical formulationis administered. An amount of from about 10³ to about 10⁷ virusparticles, and preferably about 10⁴ to 10⁶ virus particles per dose issuitable, depending upon the age and species of the subject beingtreated, and the immunogen against which the immune response is desired.Subjects and immunogens are as described above.

The present invention further provides a method of delivering anucleotide sequence into a cell. For in vitro methods, the virus can beadministered to the cell by standard viral transduction methods, as areknown in the art. Preferably, the virus particles are added to the cellsat the appropriate multiplicity of infection according to standardtransduction methods appropriate for the particular target cells. Titersof virus to administer can vary, depending upon the target cell type andthe particular virus vector, and can be determined by those of skill inthe art without undue experimentation. Preferably, at least about 1000infectious units, more preferably at least about 10,000 infectiousunits, are administered to the cell. Alternatively, administration of anadenovirus vector of the present invention can be accomplished by anyother means known in the art, as described hereinbelow.

In one preferred embodiment, the present invention provides a method ofdelivering a heterologous nucleotide sequence into a cell in vitro or invivo. According to this method a cell is infected with at least onedeleted adenovirus vector according to the present invention (asdescribed in detail hereinabove). The cell may be infected with theadenovirus vector by the natural process of viral transduction.Alternatively, the vector may be introduced into the cell by any othermethod known in the art. For example, the cell may be contacted with atargeted adenovirus vector (as described below) and taken up by analternate mechanism, e.g., by receptor-mediated endocytosis. As anotherexample the vector may be targeted to an internalizing cell-surfaceprotein using an antibody or other binding protein.

The cell to be administered the inventive virus vectors can be of anytype, including but not limited to neuronal cells (including cells ofthe peripheral and central nervous systems), retinal cells, epithelialcells (including dermal, gut, respiratory, bladder and breast tissueepithelium), muscle cells (including cardiac, smooth muscle, skeletalmuscle, and diaphragm muscle), pancreatic cells (including islet cells),hepatic cells (e.g., parenchyma), fibroblasts, endothelial cells, germcells, lung cells (including bronchial cells and alveolar cells),prostate cells, and the like. Moreover, the cells can be from anyspecies of origin, as indicated above. Preferred are cells that arenaturally transduced by adenoviruses.

The adenovirus vectors of the invention may be employed to producedproteins or peptides of interest by cells in vitro. The adenovirusencodes a heterologous nucleotide sequencers) that may encode anyprotein or peptide of interest, as described hereinabove. The nucleotidesequence preferably encodes a therapeutic protein or peptide. In morepreferred embodiments, the heterologous nucleotide sequence encodes aGAA, more preferably hGAA, which may be isolated from the cells usingstandard techniques and administered to subjects with GAA deficiencyusing enzyme replacement protocols (see, e.g., Van der Ploeg et al.,(1991) J. Clin. Invest. 87:513).

Alternatively, adenovirus vectors can be targeted to cells, includingcells that are not normally competent for transduction by adenovirusesusing antibodies, e.g., as described in U.S. Pat. No. 5,861,156 toGeorge et al.; U.S. Pat. No., 5,521,291 to Curiel et al., thedisclosures of which are incorporated herein in their entirety byreference. Alternatively, adenoviruses can be targeted to cell-surfaceproteins (e.g., receptors) by expressing a binding protein or ligand onthe surface of the adenovirus, e.g., as described by Douglas et al.,(1996) Nature Biotechnology 14:1574; U.S. Pat. No. 5,770,442 to Wickhamet al.; and U.S. Pat. No. 5,712,136 to Wickham et al. (the disclosuresof which are all incorporated herein in their entirety).

In particular embodiments of the invention, cells are removed from asubject (e.g., dendritic cells, hepatic cells, cells of the centralnervous system, myoblasts (including skeletal myoblasts), stems cells(including bone marrow cells), pancreatic cells), the adenovirus vectoris introduced therein, and the cells are then replaced back into thesubject. Methods of removing cells from subjects for treatment ex vivo,followed by introduction back into the subject are known in the art. Inone exemplary embodiment, liver cells are removed from a subject, aninventive adenovirus expressing GAA (e.g., hGAA) is introduced therein,and the liver cell expressing the heterologous GAA gene is re-introducedback into the subject. As a further alternative, the cells that aremanipulated and then introduced into the subject are provided fromanother subject or cell line.

A further aspect of the invention is a method of treating subjects invivo with the inventive virus particles. Administration of theadenovirus particles of the present invention to a human subject or ananimal in need thereof can be by any means known in the art foradministering virus vectors. Preferably, at least about 1000, morepreferably, at least about 10,000 infectious units are administered tothe subject per treatment. Preferably, the subject is a mammaliansubject, more preferably a human subject. Also preferred are subjectsthat have been diagnosed with a lysosomal storage disease or a glycogenstorage disease. More preferred are subjects who have been diagnosedwith GAA deficiency.

Type II GSD is an autosomal recessive genetic disorder that results inthe loss of activity of GAA. This disease is caused by mutations in theGAA gene itself, and results in the accumulation of glycogen in thelysosomes of skeletal and cardiac muscles resulting in cardiomyopathyand skeletal myopathy. This condition results in death in the congenital(infant-onset) form and severe debilitation in the juvenile and adultonset forms.

In patients with Type II GSD (commonly called “Pompe disease”, althoughthis term formally refers to the infantile onset form of the disease) aeficiency of acid α-glucosidase causes massive accumulation of glycogenin lysosomes disrupting cellular function (Hirschhorn, in The Metabolicand Molecular Basis of Inherited Disease, 7th ed., Vol. 2 (eds, Scriver,C. R. et al.) 2443-2464 (McGraw-Hill, N.Y., 1995). In the most commoninfantile form, patients exhibit progressive muscle degeneration andcardiomyopathy and die before two years of age. Intravenous injection ofenzyme obtained from human placenta or Aspergillus niger correctedenzyme and glycogen levels in liver but not in muscle or heart inpatients with GAA deficiency (Hug et al., (1968) Clin. Res. 16:345; deBarsy et al., in Enzyme Replacement Therapy in Lysosomal StorageDiseases (eds Tager, J. M., Hooghwinkel, G. J. M. & Daems, W. T.)277-279 (North-Holland Publishing Co., Amsterdam, 1974); Hug et al.,(1967) Cell Biol. 35:C1).

GAA may be targeted to lysosomes via the mannose-6-phosphate receptor aswell as by sequences associated with delayed cleavage of the signalpeptide (in The Metabolic and Molecular Basis of Inherited Disease, 7thed., Vol. 2 (eds, Scriver, C. R. et al.) 2443-2464 (McGraw-Hill, N.Y.,1995). Mannose-6-phosphate containing GAA enzyme from bovine testes,human urine, or medium of transiently transfected COS cells is taken upefficiently by cultured patient cells through the mannose-6-phosphatereceptor (Hoefsloot et al., (1990) Biochem. J. 272:485; Oude-Elferink etal., (1984) Eur. J. Biochem. 139:489; Reuser et al., (1984) Exp. CellRes. 155:178; Van der Ploeg et al., (1988) J. Neurol. 235:392). Bovinetestes enzyme injected intravenously in mice is targeted to thesetissues via the abundant mannose-6-phosphate receptor in heart andmuscle tissue (Van der Ploeg et al., (1991) J. Clin. Invest. 875:513).

For therapeutic use in humans, interspecies antigenicity requires theuse of human enzyme (Hug et al., (1968) Clin. Res. 16:345), but the lowabundance of the enzyme in human urine makes this source impractical(Oude-Elferink et al. (1984) Eur. J. Biochem. 139:489), making genetherapy approaches desirable.

Gene therapy is a particularly appropriate approach to treating GAAdeficiency, in that recombinant human acid α-glucosidase made inbacteria was not catalytically active (Martiniuk et al. (1992) DNA andCell Biology 11:701 ), and that recombinant enzyme made usingbaculovirus in insect cells was active, but it was not taken upefficiently by human fibroblasts (Wu et al., (1993) Am. J. Hum. Genet.53(Supplement):963). In contrast, enzyme secreted by transientlytransfected COS cells was active and taken up efficiently by fibroblasts(Hoefsloot et al., (1990) Biochem. J. 272:485).

Thus, it appears that the required post-translational modifications makeit difficult to produce the enzyme in inexpensive expression systems(e.g., bacterial and insect systems) in a form where it is both activeand capable of being taken up by the desired tissues. This complicatesthe manufacture of active, correctly modified enzyme outside of theorganism to be treated. Introduction of the genetic material into thepatient allows the synthesis to be performed in the cells of thatorganism and eliminates these difficulties.

Accordingly, a further aspect of the present invention is a method oftreating a subject with GAA deficiency, including infantile (Pompedisease), juvenile and adult-onset forms of the disease. Preferably, thesubject is a human subject. According to this method, the subject isadministered a biologically-effective amount of a nucleotide sequenceencoding a GAA protein to a non-muscle tissue of the subject.Preferably, the non-muscle tissue is an organ tissue (e.g., brain,pancreas, liver), most preferably, the liver. Nucleotide sequencesencoding GAA are as described hereinabove, and include nucleotidesequences encoding the mature and precursor GAA protein. A“biologically-effective” amount of the nucleotide sequence is an amountthat is sufficient to result in uptake and expression of the nucleotidesequence by at least one cell in the target tissue or organ. Preferably,at least about 10% of the target cells take up and express thenucleotide sequence encoding the GAA protein, more preferably at leastabout 25%, 50%, 75%, 90%, 95%, 99% or more of the target cells take upand express the nucleotide sequence. In still more preferredembodiments, essentially all of the target cells take up and express thenucleotide sequence encoding the GAA protein. In preferred embodiments,the nucleotide sequence encoding the GAA is administered to the liver.Modes of administration to the liver are as described hereinbelow.

The nucleotide sequence encoding the GAA can be administered by anymethod known in the art, including viral vectors, liposomes, direct DNAinjection, and the like. Preferably, the nucleotide sequence is carriedby a recombinant deleted adenovirus vector of the present invention (asdescribed hereinabove).

Preferably, the cells (e.g., liver cells) take up the nucleotidesequence encoding the GAA protein, express the GAA protein, and secreteit into the circulatory system, where it is delivered to target tissues(e.g., muscle) in a therapeutic amount. By a “therapeutic amount” it isintended that the GAA is taken up by the target tissue and alleviates(i.e., decreases, mitigates, reduces) at least one of the symptoms ofGAA deficiency in the subject. It is not necessary that the symptoms ofGAA deficiency be eliminated, as long as the benefits outweigh thedetriments of delivering the GAA to the target tissue.

The present inventors have found that it is advantageous to express the110 kD precursor form of the GAA protein, containing mannose-6-phosphateresidues. While not wishing to be bound by any particular theory of theinvention, it appears that the GAA precursor is taken up bymannose-6-phosphate receptors on the surface of target tissues, and theinternalized precursor protein is processed to its mature form, whichthen produces a therapeutic effect on the target tissue.

In practicing this embodiment of the invention, high-level expression ofthe GAA (or any other protein that is normally not secreted) by theliver (or other organ) is advantageous in that it appears that thenatural mechanisms within the hepatocyte that target the GAA protein tothe lysosomal compartment are saturated, thereby resulting in the“excess” protein entering the secretory pathway for delivery to targettissues and organs. The inventive adenoviruses are particularly suitedto this purpose as adenoviruses are both highly infectious and producehigh levels of transgene expression.

Accordingly, these methods overcome many of the problems raised bydirect delivery of nucleotide sequences to muscle tissue. Administrationof virus vector by intramuscular injection has demonstrated that thevirus is localized to the site of injection (Tsujino et al. (1998) HumanGene Therapy 9:1609 Nicolino et al., (1998) Human Molecular Genetics7:1695; Pauly et al., (1998) Gene Therapy 5:473; Zaretsky et al., (1997)Hum. Gene Ther. 8:1555). Accordingly, to deliver a GAA transgene toevery muscle would require multiple injections directly into each of themuscle groups in Type II GSD patients. Moreover, after intravenousadministration, adenovirus vectors normally transduce only a smallnumber of muscle fibers (Stratford-Perricaudet et al., (1992). ClinInvest. 90:626; Kass-Eisler et al., (1994) Gene Ther. 1:395). Thepresent inventors have found that expression of GAA from the liver aftera single intravenous injection advantageously circumvents the problem ofdirectly transducing cardiac and skeletal muscle target tissues.

Those skilled in the art will understand that the method describedhereinabove can be used to produce any protein product in one tissue ororgan and deliver it to another target tissue, e.g., via the circulatorysystem. Preferably, this method is used to produce other proteins orpeptides associated with metabolic diseases, more preferably, lysosomaland glycogen storage diseases, each as described above. Also preferredare methods of producing proteins or peptides for delivery to muscletissue. Further preferred are methods wherein the nucleotide sequence isintroduced into the liver for expression and delivery to other targettissues.

Likewise, the present invention provides a method of producinghigh-level expression of other lysosomal proteins by the liver (or otherorgans and tissues). The protein can be expressed and secreted by theliver, and delivered to target tissues by the systemic circulation,where it is taken up by mannose-6-phosphate receptors on target tissues.

Alternatively, the present invention provides a method of treating asubject with GAA deficiency comprising administering to the subject atherapeutically-effective amount of the inventive deleted adenovirusparticles (as described hereinabove) carrying a heterologous nucleotidesequence encoding a GAA (as described hereinabove). A“therapeutically-effective” amount as used herein is an amount ofadenovirus that is sufficient to alleviate (e.g., mitigate, decrease,reduce) at least one of the symptoms associated with GAA deficiency. Itis not necessary that the GAA eliminate the symptoms of GAA deficiency,as long as the benefits outweigh the detriments of GAA administration.

Exemplary modes of administration include oral, rectal, transmucosal,topical, transdermal, inhalation, parenteral (e.g., intravenous,subcutaneous, intradermal, intramuscular, and intraarticular)administration, and the like, as well as direct tissue (e.g., muscle) ororgan injection (e.g., into the liver, into the brain for delivery tothe central nervous system), alternatively, intrathecal, directintramuscular, intraventricular, intravenous, intraperitoneal,intranasal, or intraocular injections. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Alternatively, one may administer the virus in a localrather than systemic manner, for example, in a depot orsustained-release formulation.

The adenovirus vectors disclosed herein may alternatively beadministered to the lungs of a subject by any suitable means, but arepreferably administered by administering an aerosol suspension ofrespirable particles comprised of the inventive adenovirus vectors,which the subject inhales. The respirable particles may be liquid orsolid. Aerosols of liquid particles comprising the inventive adenovirusvectors may be produced by any suitable means, such as with apressure-driven aerosol nebulizer or an ultrasonic nebulizer, as isknown to those of skill in the art. See, e.g., U.S. Pat. No. 4,501,729.Aerosols of solid particles comprising the inventive virus vectors maylikewise be produced with any solid particulate medicament aerosolgenerator, by techniques known in the pharmaceutical art.

In particularly preferred embodiments of the invention, the nucleotidesequence of interest is delivered to the liver of the subject.Administration to the liver can be achieved by any method known in theart, including, but not limited to intravenous administration,intraportal administration, intrabiliary administration, intra-arterialadministration, and direct injection into the liver parenchyma.

Dosages will depend upon the mode of administration, the disease orcondition to be treated, the individual subject's condition, theparticular virus vector, and the gene to be delivered, and can bedetermined in a routine manner. Preferably, at least about 10,000infectious units of the inventive adenovirus vectors are administered tothe subject. Exemplary doses for achieving therapeutic effects are virustiters of 10⁸-10¹⁴ particles, preferably 10¹⁰-10¹³ particles, yet morepreferably 10¹² particles.

In particular embodiments of the invention, more than one administration(e.g., two, three, four, or more administrations) may be employed toachieve therapeutic levels of gene expression.

Having now described the invention, the same will be illustrated withreference to certain Examples, which are included herein forillustration purposes only, and which are not intended to be limiting ofthe invention.

EXAMPLE 1 Construction of the Ad Vector Deleted for the E2b-PolymeraseGene

The ˜20 kb Xba-BamHI subfragment of pBHG11 (Microbix, Toronto) whichcontains the Ad E2b region was subcdoned into pBluescriptKSII+(Stratagene, La Jolla, Calif.), yielding pAXB. The pAXB plasmid wasdigested with BspEI, T4 DNA polymerase end-filled, BamHI digested, andthe ˜9.0 kilobase pair (kb) fragment was isolated. Plasmid pAXB was alsodigested with BspHI, T4 DNA polymerase end-filled, BamHI digested, andthe ˜13.7 kb fragment was ligated to the previously isolated 9.0 kbfragment, generating pAXB-Δpol. This subdloning strategy deleted 608base pairs (Δpol: Ad5 nucleotide 7274-7881) within the amino terminus ofthe polymerase gene. This deletion also effectively removed ORF 9.4,present on the rightward reading strand in this region of the Ad genome.The Xba-BamHI subfragment of pAXB-Δpol was reintroduced into Xba-BamHIdigested pBHG11, to generate pBHG11-Δpol (FIG. 1, Panel A).

Theoretically, pBHG11-Δpol should have been capable of generatingrecombinant [E1-Δpol] Ad vectors after cotransfection of polymerasetranscomplementing cells with a conventional Ad shuttle plasmid (Beft,J. J., et al., (1994) PNAS USA 91:8802-8806), unfortunately we werenever able to generate such a vector with this approach. It is possiblethat our version of pBHG11 had acquired a cryptic point mutationprohibiting viable vector isolation. We therefore co-transfected thepBHG11-Δpol plasmid with Scal digested dl7001 derived genomic DNA intothe polymerase expressing cell line C-7 (FIG. 1, Panel B). The Ad dl7001genomic DNA had an ˜3.0 kb deletion within the E3 region of the Adchromosome and was isolated as an intact virion DNA-terminal proteincomplex, dl7001-TP as described in Jones et al., Cell 13:181-88 (1978).The C7 cell line has been previously described (Amalfitano, et al.,(1996) PNAS USA 93:3352-56). Briefly, these cells are capable oftranscomplementing [E1-]Ad, as well as temperature-sensitive Ad mutantsdefective in both the polymerase and preterminal protein genes. Thiscell line was accomplished by the stable cointroduction of transgenesconstitutively expressing the polymerase and preterminal protein genesinto human 293 cells.

The co-transfection strategy resulted in the isolation of two viableviruses one with the Δpol deletion in the dl7001 background (AdΔpol) andone with the Δpol deletion in the pBHG11 background, AdΔpol/pBHG11).While both viruses were viable, the dl7001 derived virus demonstratedsuperior growth characteristics and was therefore selected for furtherwork.

EXAMPLE 2 Construction of [E1-, Δpol, E3-] and [E1-, Δpol, ΔpTP, E3-] AdVectors

The AdΔpol virus was grown to high titer, and viral DNA isolated aspreviously described (Amalfitano, et al., (1996) PNAS USA 93:3352-56),digested with Ascl, T4 polymerase end-filled, and Bst1107I digested. The˜9.3 kb blunt-ended Δpol containing fragment was subcloned into theBst1107I digested shuttle plasmid pAdAscL. This subcloning strategyyielded pAdAscL-Δpol, a new shuttling plasmid specifically designed forthe rapid isolation of recombinant Ad vectors deleted for both the Ad E1and polymerase genes. The pAdAscL-Δpol plasmid contained nucleotides1-15,671 of the left end of the Ad5 genome, but was effectively deletedfor the E1 genes (Ad nt 358-3328, replaced by the Ascl site) and alsowas deleted for the 608 bp Δpol deletion. A nuclear-targeted bacterialα-galactosidase transgene (LacZ) flanked by a minimal cytomegalovirus(CMV) promoter/enhancer element, the MINX intron (Niwa, M., et al.,Genes Dev 4:1552-1559 (1990) and a simian virus 40 (SV-40) derivedpolyadenylation signal was subcloned into the Ascl site of pAdAscL-Δpol,generating the shuttle plasmid pAdCMV/LacZ/Δpol (FIG. 1, Panel C). Tenmicrograms of pAdCMV/LacZ/Δpol linearized with BspHI (restriction sitewithin 60 bp of the left end of the Ad virus) was CaPO₄-cotransfectedwith 500 ng of Xbal, Clal, and Scal digested dl7001-TP virion DNA ontothree 60 mm dishes containing 2×10⁶ Ad-polymerase expressing C-7 cells(FIG. 1, Panel C). The multiple restriction enzyme digestion of dl7001virion DNA significantly reduced the isolation of non-recombinantviruses after transfection. Sixteen hours after transfection the cellswere harvested and mixed with ˜8×10⁶ C-7 cells (non-transfected). Thecell mixture was distributed to nine, 24-well tissue culture clusterplates, and incubated at 37.0° C. for 5-9 days. Individual wellsdemonstrating viral CPE were harvested, and the isolated virus amplifiedby repeated infection of either B-6 or C-7 cells. Isolation of theAdLacZΔpol recombinant vector was subsequently confirmed by (1)α-galactosidase conversion of the chromogenic substrate X-gal in cellstransduced by the vector (2) DNA restriction mapping of the vectorgenome, and (3) by multiple functional studies.

To create Ad vectors containing deletions in both the polymerase andpreterminal protein regions, shuttle plasmid pAdCMV/LacZ/Δpp wasgenerated. The pAdCMV/LacZ/Δpol shuttle plasmid was digested with BspE1(releasing a ˜2.0 kb subfragment encoding the 3′ end of the pTP gene). Apreviously-modified BspE1 subfragment of the pTP gene that contained adeletion within the pTP coding region was ligated into the BspE1-digested plasmid. This strategy resulted in the isolate of the shuttleplasmid pAdCMV/LacZ/Δpp, which was similar to pAdCMV/LacZ/Δpol exceptthat it also contained a 433 bp deletion within the 3′ end of the pTPgene. The deletion spanned nucleotides 9198 to 9630 of the Ad5 genome(deletion confirmed by sequencing). The deletions spanned the 3′ portionof the pTP gene that encodes the critical serine residue required forpTP binding to the 5′ end of the Ad genome, as well as other criticalpTP functions.

Ten μg of pAdCMV/LacZ/Δpp was linearized with BspHI (restriction sitewithin 60 bp of the left end of the Ad virus), was CaPO₄-cotransfectedinto C-7 cells with 500 ng of Xbal, Clal, and Scal digested dl7001virion DNA, and incubated overnight. The transfected cells weresubsequently distributed to ten 24-well dishes, allowing for clonalisolation of recombinant vectors. Several wells were found to containrecombinant vectors encoding β-galactosidase and simultaneously deletedfor E1, E3, pol and pTP. Isolation of the multiply-deleted, AdLacZ/Δpprecombinant vector was also subsequently confirmed by (1)β-galactosidase conversion of the chromogenic substrate X-gal in cellstransduced by the vector; (2) DNA restriction mapping of the vectorgenome; and (3) by multiple functional studies.

EXAMPLE 3 AdΔpol, AdLacZΔpol and AdLacZΔpp Vector Genome-ReplicationStudies

The respective cell lines were infected at the multiplicity of infection(MOI) indicated in FIGS. 2 and 3 with either AdΔpol, Adsub360LacZ (E1deletion alone), AdLacZΔpol (E1 deletion+polymerase deletion) orAdLacZΔpp (E1 deletion, polymerase deletion, and pTP gene deletion), andincubated at 37.0° C. for the indicated times. Infected cells were thenharvested and DNA prepared and analyzed as previously described(Amalfitano, et al., (1996) PNAS USA 93:3352-56). The results in FIGS. 2and 3 show that even in the presence of high levels of E1 activity theΔpol modification conveys upon the vector a severe replication blockade.This block is significantly greater than that displayed by thefirst-generation Adsub360LacZ vector (FIG. 3). FIGS. 2 and 3 also show,in contrast to the reports using so-called “gutted” vectors which areessentially undetectable by 12-24 hours post infection, the inputgenomes persist to at least 75% of input virus levels at 24 hours and atleast 50% at 48 hours.

EXAMPLE 4 Ad Vector Kinetics and One Stop Burst Assays

Kinetics Assay: Tissue culture dishes (60 mm) containing 2×10⁶ 293 cells(express E1 protein), B6 cells (express E1 and polymerase proteins), orC-7 cells (express E1, polymerase, and pTP proteins) were infected withAdLacZΔpol at an MOI of 0.01 β-galactosidase forming units (BFU) percell or with AdLacZΔpp at an MOI of 0.5 β-galactosidase forming units(BFU) per cell. Cells and media were harvested from the dishes afterincubation at 37.0° C. for the times indicated in FIGS. 4 and 5. Thenumber of BFU produced was then determined by limiting dilutioninfection of C-7 cells or LP-293 cells. Eighteen hours later infectedcells were stained for β-galactosidase activity, and the number oftransducing particles was quantified by visual inspection of bluestaining cells. The BFU generated in the original lysate was thendetermined by multiplying the number of stained nuclei by theappropriate dilution. An identical infection of 293 cells withAdsub360LacZ was included to compare the kinetics of growth of theAdLacZΔpp vector to the first-generation, Adsub360LacZ vector. Resultsare shown in FIGS. 4 and 5.

One Step Burst Assay: 2×10⁶ 293 or B-6 or C-7 cells were respectivelyinfected with AdLacZΔpol, Adsub360LacZ or AdLacZΔpp (in triplicate) atan MOI of 5, incubated at 37.0° C. for 40 hours (Adsub360LacZ infectionof 293 cells and AdLacZΔpol of B-6 cells) or 60 hours (AdLacZΔppinfection of C-7 cells), and the total BFU generated was determined bylimiting dilution assay, as previously described for the kinetic assays.

EXAMPLE 5 Ad Vector Late Gene-Expression Studies

LP-293 cells, B-6 cells, or HeLa cells were infected with either AdΔpol,Adsub360LacZ, AdLacZΔpol or AdLacZΔpp at the indicated MOIs in FIGS. 6,7 and 8 and incubated at 37.0° C. for 40-48 hours. Infected cells wereharvested, rinsed with PBS, and lysed in Tris Cl (pH 6.8), 4%sodium-dodecyl sulfate, 10% glycerol, and 10% B-mercaptoethanol. Proteinextracts were freeze thawed three times, DNA sheared, and proteinconcentrations determined via the Bradford assay utilizing the CoomassiePlus staining reagent (Pierce, Rockford, Ill.). Equivalent amounts ofeach protein lysate were heated to 100.0° C. for two minutes andelectrophoretically separated in a 10% SDS-polyacrylamide gel. Theseparated proteins were wet-transferred to a Biotrace NT membrane(Gelman Sciences, Ann Arbor, Mich.) and probed with a rabbit polyclonalantibody (supplied by R. Gerard, University of Texas Southwestern,Dallas, Tex. generated against the knob portion of the 66 kD Ad-fiberprotein monomer. Bound antibody was detected with the ECL detectionsystem (Amersham Life Sciences, Arlington Heights, Ill.).

EXAMPLE 6 In vivo Administration of AdLacZΔpol

Sixty 150 mm tissue culture plates containing ˜2.5×10⁷ C-7 cells wereinfected with the AdLacZΔpol virus at an approximate MOI of 5, andincubated at 37.0° C. for 40 hours. The infected cells were harvested,resuspended in 10 mm Tris Cl (pH 8.0), sonicated, and the virus purifiedby two rounds of cesium chloride density centrifugation. The viralcontaining band was desalted over a Sephadex CL-6B column (PharmaciaBiotech, Piscataway, N.J.), glycerol added to a concentration of 12%,and aliquots were stored at ˜80° C. The titer of this stock was 6×10¹⁰BFU per ml. The total number of particles in this stock was 1.2×10¹², asdetermined by measurement of the O.D. 260 of an aliquot of the virusafter SDS-lysis (Mittereder et al., (1996) J. Virol. 70:7498), thereforethe bioactivity of the preparation was at a minimum of 0.05,(6×10¹⁰/1.2×10¹²) a value similar to that achieved after isolation offirst-generation Ad vectors (Id.). Seven to nine week old BALB/c micewere injected in the left tibialis anterior muscle or via the tail veinwith a PBS solution containing 1×10⁹ BFU of AdLacZΔpol. Five-six daysafter infection, the mice were sacrificed, and the muscle or liverspecimens removed and frozen in OCT Compound. Cryosections wereobtained, briefly fixed in a 3.7% formaldehyde/PBS solution, stainedovernight for β-galactosidase activity, and rinsed in PBS and brieflypost-fixed in 3.7% formaldehyde, 0.5% glutaraldehyde in PBS. Sectionswere then eosin counterstained and photographed. Results are shown inFIG. 9 and as described in Example 11.

EXAMPLE 7 ΔPol Vectors

The isolation of packaging cell lines that co-express the Ad E1 andpolymerase genes, based upon their ability to support the growth ofAd-polymerase temperature sensitive (ts)-mutants has previously beendescribed (Amalfitano et al., (1996) Proc. Natl. Acad. Sci. USA93:3352-3356). The virus AdΔpol, constructed as described herein and asshown in FIG. 1, contains a 608 bp deletion within the polymerase gene(FIG. 1, Panel B). The Δpol deletion described in Example 1 alsoeffectively deleted ORF 9.4, with no consequence in regard to the growthpotential of the resultant viruses. Confirmation of the AdΔpol genomicstructure was accomplished by restriction enzyme digestion (FIG. 10).AdΔpol had a severe replication defect when not grown in cellsexpressing the Ad polymerase, confirming the lack of polymerase activitysecondary to the introduced 608 bp deletion (FIG. 4). Despite highlevels of E1 activity (both from the AdΔpol genome as well as the E1expressing 293 cells) AdΔpol is incapable of significant replication incells other than the C7 cell line. This contrasts with the behavior offirst generation Ad vectors in vitro and in vivo which are significantlyleaky even in presumably non-permissive hosts and target cells (Lieber,A. et al., (1996) Journal of Virology 70:8944-8960, Yang, Y, et al.,(1994) Nat Genet 7:362-369).

EXAMPLE 8 Isolation and Growth Kinetics of AdLacZΔpol

To facilitate the production of [E1-, E3-, Δpol] Ad vectors, a shuttlingsystem was engineered for their construction (FIG. 1, Panel C).Co-transfection of linearized pAdCMV/LacZ/Δpol with multiply digesteddl7001 DNA-TP complex resulted in the successful isolation ofAdLacZΔpol. The genomic structure of AdLacZΔpol was confirmed byrestriction enzyme analysis (FIG. 11). The kinetics of AdLacZΔpol growthwas then determined in 293 cells and B-6 cells (FIG. 12). Note thedramatic lack of production of infectious AdLacZΔpol in LP-293 cells,despite the presence of high levels of E1 activity in this cell line.Furthermore, one step burst assays of B-6 cells infected with AdLacZΔpolor Adsub360LacZ clearly demonstrated that the AdLacZΔpol vector could beproduced to as high (or higher) a titer as the Adsub360LacZ vector. Forexample, when 2×10⁶ B-6 cells were infected at an MOI of 5 BFU withAdLaCZΔpol or Adsub360LacZ (each in triplicate) the total BFU releasedafter a 40 hour infection was 1.18×10⁸±4.2×10⁷ BFU for AdLacZΔpol, and1.78×10⁷+8.9×10⁶ BFU for Adsub360LacZ. High titer growth of theAdLacZΔpol vector in the B-6 cell line is a significant attribute of thepresent invention, not only for lower cost and higher efficiencyclinical grade production, but also because previously describedpackaging cell lines designed to allow the growth of modified Ad vectorsare sometimes inefficient, likely due to the toxicity of the coexpressedAd genes (Zhou, H. S., et al., (1996) Journal of Virology 70:7030-7038).

EXAMPLE 9 AdLacZΔpol is Blocked in Replication

Infection of B-6 cells allowed high level replication of the AdLacZΔpolgenomes; however, an identical infection of 293 cells demonstrated adramatic block of replication (FIG. 2). This result confirmed that, evenin the presence of high levels of E1 activity, the Δpol modification ofthe present invention conveys upon the vector a severe replicationblockade. Despite a lack of significant replication, the AdΔpol andAdLacZΔpol genomes were still present at near input levels 24 hourspost-infection in 293 cells, and decreased only after 48 hourspost-infection. Supporting these observations, high titer infection ofHeLa cells (lacking both E1 and polymerase activities) with AdLacZΔpoldemonstrated a significantly greater replication block than displayed bythe first generation Adsub360LacZ vector (FIG. 3).

Despite the lack of replication demonstrated above, the AdLacZΔpolpersisted to at least 75% of input virus levels within 24 hours of HeLacell infection, and dropped to 50% of input virus by 48 hours afterinfection (FIG. 3). This result is in contrast to a report utilizing“gufted Ad vectors” that are devoid of much of the Ad genome (Lieber, etal., (1996) J. Virol. 70:8944-60). In the latter report, 50% of the“gutted Ad vector” genomes were degraded within 5 hours of transductionof cells both in vitro and in vivo, and was essentially undetectable by12-24 hours post transduction. Id. Consequently, unlike earliermassively deleted “gutted” Ad vectors, Δpol Ad vectors of the presentinvention severely blocked in their ability to replicate (even in thepresence of excessive levels of E1 activity), but unlike gutted Advectors, this blockade does not simultaneously result in a rapid lossdestabilization) of their genomes.

EXAMPLE 10 AdLacZΔpol Late Gene Expression is Blocked inNoncomplementing Cell Lines

Cell types 293 and B-6 were infected with AdLacZΔpol and assessed forviral late gene expression, as determined by fiber protein accumulation.As shown in FIG. 6, there is at least a 10,000 fold reduction in theability of the AdLacZΔpol vector to produce the fiber protein afterinfection of 293 cells, in contrast to infection of thepolymerase-complementing B-6 cell line. Further, HeLa cells wheninfected with the AdLacZΔpol virus do not produce detectable fiberprotein (FIG. 7). In contrast, fiber expression is readily detectedafter infection of HeLa cells with the Adsub360LacZ. Together, theseresults demonstrated another benefit of the AdLacZΔpol vector, asignificantly decreased expression of viral late genes, secondary to thesevere replication blockade afforded by the presence of the Δpol in themodified vector. Ad late gene products such as the fiber protein arepotent antigenic epitopes in vivo, therefore decreased expression of thelate, E1, and polymerase gene products may result in a greater efficacyfor AdΔpol vectors in vivo.

EXAMPLE 11 In vivo Transduction with the AdLacZΔpol Vector

1×10⁹ BFU of AdLacZΔpol were injected intravenously (for livertransduction) or into the left tibialis anterior muscle of 7-9 week oldBALBIC mice. Five to six days later the respective tissues were excisedand stained for β-galactosidase activity. As demonstrated in FIG. 9, theAdLacZΔpol vector is capable of extensive transduction and expression ofthe bacterial β-galactosidase gene in liver tissues. The same result wasachieved after intramuscular administration of AdLacZΔpol. Therefore,despite the additional replication blockade provided by the deletion ofboth the E1 and polymerase genes in the AdLacZΔpol vector, efficienttransduction and transgene expression occurred in these tissues. Thisagain is in contrast to some recently described helper virus-dependentAd vector systems, whose modified mini-genomes were rapidly eliminatedin vivo, before significant transgene expression occurred (Lieber, A. etal., (1996) Journal of Virology 70:8944-8960).

EXAMPLE 12 Construction of Multiply-Deleted ΔpTP Ad Vectors

The construction of the pAdCMV/LacZ/Δpol shuttling plasmid and the E1,E3, and polymerase deleted vector AdLacZΔpol has been described inExample 1 and Example 2. To create a shuttle plasmid with deletions inthe preterminal protein region, the pAdCMV/LacZ/Δpol shuttle plasmid isdigested with BspE1 (releasing a ˜2.0 kb subfragment encoding the 3′ endof the pTP gene) and reinserted into a previously modified BspE1subfragment that contains a deletion within the pTP coding region. Theresulting shuttling plasmid pAdCMV/LacZ/Δpp was isolated. This plasmidis similar to pAdCMV/LacZΔpol, except that it also contained a 433 bpdeletion within the 3′ end of the pTP gene (FIG. 13). The 433 bp pTPdeletion spans nucleotides 9198-9630 of the Ad5 genome. This fact wasconfirmed by sequencing across the deletion. Creation of thepAdCMV/LacZΔpp resulted in the deletion of a significant portion of thepTP coding region, and spanned the 3′ portion of the pTP gene thatencodes the critical serine residue required for pTP binding to the 5′end of the Ad genome, as well as other critical pTP functions (SchaackJ, et al., (1990) Genes Dev 4:1197-208, Webster A, et al., (1997) Joumalof Virology;71:6381-9).

Ten micrograms of pAdCMV/LacZ/Δpp linearized with BspHI (restrictionsite within 60 bp of the left end of the Ad virus) wasCaPO₄-cotransfected into C-7 cells with 500 ng of Xbal, Clal, and Scaldigested dl7001 virion DNA, and processed as described in Example 2above. Isolation of the multiply deleted, AdLacZΔpp recombinant vectorwas subsequently confirmed by 1) β-galactosidase conversion of thechromogenic substrate X-gal in cells transduced by the vector 2) DNArestriction mapping of the vector genome, and 3) by multiple functionalstudies (see below).

In order to grow the multiply-deleted Ad vectors of the presentinvention, a packaging cell line is required which can transcomplementall of the essential gene functions deleted in the vectors. We havedemonstrated that the B-6 and C-7 cell lines, isolated and characterizedfor their ability to complement temperature sensitive polymerase (B-6and C-7) and pTP mutants (C-7) (Amalfitano, A., et al., (1996) PNAS USA93:3352-3356) also complement polymerase, IVa2 and/or pTP deletionmutants.

EXAMPLE 13 Transcomplementation of Δpp Ad Vectors by C-7 Cells

The present investigations have found that ΔTP and Δpol Ad vectors canbe packaged when grown in cell lines selected for their ability tosupport the growth of adenovirus strains with temperature sensitive (ts)mutations within the polymerase or pTP genes, e.g., the C-7 cell line(Amalfitano et al., (1997) Gene Ther. 4:258).

Ad vectors that simultaneously lack the Ad E1, E3, polymerase, and pTPgene activities are capable of high level growth on such strains, suchas the preferred C-7 strain. A shuttling system was developed tofacilitate the production of a multiply deleted Ad vectors. Targetedhomologous recombination after co-transfection of the linearizedpAdCMV/LacZ/Δpp shuttle plasmid with multiply digested dl7001 DNAresulted in the successful generation of a unique Ad vectorsimultaneously deleted for the E1, E3, polymerase, and pTP geneactivities, referred to as AdLacZΔpp (FIG. 13). Restriction enzymeanalysis was utilized to confirm the genomic structure of AdLacZpp (FIG.14). Note that in comparison to the AdLacZΔpol vector (deleted forportions of the E1, E3, and polymerase genes), the AdLacZΔpp vectorgenome contains an additional 433 bp deletion, that represented thelocation of the pTP-specific deletion (FIG. 15). The deletion within thepTP open reading frame eliminates the critical portion of the proteinabsolutely required for normal function, and completely inactivates pTPactivities in resultant vectors containing this deletion.

To demonstrate this inactivation, the growth kinetics of AdLacZΔpp wereevaluated when transcomplementation was attempted in 293, B-6, or C-7cells (FIG. 5). Despite the presence of high levels of E1 activity inthe 293 cell line, or of both E1 and polymerase gene activities in theB-6 cell lines, no infectious AdLacZΔpp was produced after infection ofeither cell line, even after prolonged incubation times. This resultdemonstrated that polymerase and pTP gene functions were disabled in theAdLacZΔpp vector, and that this blockade could only be relieved when thevector was grown in the multiply-transcomplementing C-7 cell line. Thekinetics of growth of AdLacZΔpp in C-7 cells was slightly delayed whencompared to the growth of the first generation vector Adsub360LacZ in293 cells, although final titers were only slightly reduced compared tothe first generation vector. For example, one step burst assays of C-7cells infected with AdLacZΔpp at an MOI of 5 produced a titer of1.0×10⁸±3.3×10⁷ BFU (n=3) as compared to Adsub360LacZ vector titerproduction of 2.0×10⁸±3.7×10⁷ BFU in 293 cells (n=3). High titer growthof the AdLacZΔpp vector in the C-7 packaging cell line was extremelysignificant. This result demonstrates that the vectors of the presentinvention can be produced to high titer without the need for acontaminating, transcomplementing helper virus.

Infection of 293 or B-6 cells with AdLacZΔpp demonstrated that the Δppdeletion introduced a dramatic block to replication of the AdLacZΔppgenome in the respective cell lines (FIG. 16). However, an identicalinfection of C-7 cells allowed high level replication of the AdLacZΔppgenome. The example demonstrates that the Δpp deletion conveyed upon thevector a severe replication blockade, even in the presence of E1activity in 293 cells, or of both E1 and polymerase activities in B-6cells. This is in contrast to previously reported studies with the tspTP mutant sub-100, which is temperature sensitive due to an in-frameamino acid addition within the amino-terminus of the pTP protein(Amalfitano A, et al., (1997) Gene Ther., 4:258). In this previousstudy, the sub-100 mutant demonstrated a replication defect that waseffectively transcomplemented by the presence of the polymerase protein.Id. Clearly, the presence of the 433 bp pTP deletion significantlyinhibited AdLacZΔpp genome replication, due to complete elimination ofcritical pTP functions. In addition, the input AdLacZΔpp genomesremained present at near input levels 24 hours post infection in 293 orB-6 cells. The same result was also confirmed after infection of HeLacells (lacking E1, polymerase, and pTP activities) with AdLacZΔpp (FIG.17). In contrast to the significant replication observed after infectionof HeLa cells with the first generation vector Adsub360LacZ, theAdLacZΔpp input genomes failed to replicate and persisted at near inputlevels for up to 48 hours after HeLa cell infection (FIG. 17). Thisresult demonstrates that absence of pTP gene activities (and a completelack of replication) in the multiply-deleted Ad vectors of the presentinvention did not result in a destabilization and/or a rapid loss of themultiply-deleted vector genomes.

EXAMPLE 14 ΔTP Deletions Prevent Ad Late Gene Expression

Many DNA viruses (polyoma, simian virus-40, Ad) have life cycles thatare highlighted by early and late phases. Usually the early phasereflects the expression of viral genes required for replication of theviral genome. The late phase reflects the high level expression of viralstructural proteins after viral genome replication has commenced. Foradenovirus, the expression of the late genes is dependent on thephysical replication of the genome, an event that acts in cis toactivate the viral major late promoter (MLP) (Thomas et al., (1980) Cell22:523).

The results described herein demonstrate that the ΔTP deletion mutantalso prevent Ad late gene expression. The late gene expression blockadewas demonstrated after HeLa cells were infected with the AdLacZΔppvector (FIG. 8). Fiber protein expression (fiber mRNA transcripts arederived from initiation at the MLP) was readily detected after infectionof HeLa cells with the Adsub360LacZ vector, previously shown to becapable of replication in 293 cells. In contrast, infection of HeLacells with the AdLacZΔpp vector did not result in detectable fiberexpression.

EXAMPLE 15 In vivo Administration of AdLacZΔpp

Sixty, 150 mm tissue culture plates containing ˜2.5×10⁷ C-7 cells wereinfected with the AdLacZΔpp virus at an approximate MOI of 5, andincubated at 37.0° C. for 48 hours. Virus from the infected cells wasisolated by CsCl₂ banding as described in Example 11. Amounts ofinfectious vector obtained were essentially equivalent to that obtainedwith first generation Ad vectors. The concentrated virus was desaltedafter overnight dialysis in phosphate-buffered saline (PBS) at 4.0° C.,glycerol added, and aliquots stored at −20° C. Seven to nine week oldsevere-combined immune deficient (SCID) or C57BI/6 mice were injectedintravenously with a PBS solution (typically 150-200 uL) containing4×10⁹ BFU of the respective vectors. All animal experiments were carriedout as previously approved by the Duke University Animal Care and UseCommittee. Three days after infection, the mice were sacrificed, andliver specimens were frozen in OCT Compound. Cryosections were obtained,briefly fixed in a 3.7% glutaraldehyde/PBS solution, stained overnightfor β-galactosidase activity, and photographed, as described in Example11.

EXAMPLE 16 Results of in vivo Administration of AdLacZΔpp

The multiply deleted Ad vectors of the present invention are capable ofin vivo transduction with modified Ad vectors. 4×10⁹ β-galactosidaseforming units (BFUs) of the AdLacZΔpp vector were respectively injectedintravenously (for liver transduction) into 7-9 week old severe combinedimmune deficient (SCID) or C57BI/6 female mice. Similar injections werecarried out with the first-generation vector Adsub360LacZ, and withAdLacZΔpol. Three days later liver tissues were excised and stained forβ-galactosidase activity. As demonstrated in FIG. 18, the AdLacZΔppvector is capable of extensive transduction and expression of thebacterial β-galactosidase gene in liver tissues of injected SCID mice(also in C57BI/6 mice, data not shown). The level of transduction wasessentially equivalent to that demonstrated with the use of other, lessextensively modified vectors (FIG. 18). Therefore, efficienttransduction and transgene expression occurred in hepatic tissues usingvectors according to the present invention despite the deletion of theE1, polymerase and pTP genes in the AdLacZΔpp vector.

The inability of first-generation Ad vectors to persist aftertransduction of immune-competent hosts has been the major barrier to Admediated gene therapy paradigms. We have analyzed a unique Ad vector inan hepatic model of neoantigen transduction utilizing immune-competentmice. Hepatic gene transfer of bacterial β-galactosidase via an Advector deleted for both E1 and polymerase activities resulted inextended persistence of the vector genome to greater than two months(experiment duration). In comparison, use of a traditional [E1-] Advector encoding the same transgene resulted in a rapid loss of alltransduced cells within I month of transduction. The extendedpersistence of the modified vector was substantiated by a number ofobservations that included: i) extended durations (>1 month) oftransduced bacterial {overscore (U)}-galactosidase enzyme activities in75-100% of the hepatocytes, ii) an extended duration of transcription(>1 month) from the transgene, and iii) the extended persistence ofsignificant amounts of the vector genome (4.4 vector genomes/hepatocyte)at 28 and 56 dpi. In addition, utilization of the modified vectorsignificantly decreased the hepatotoxicity usually associated withhepatic transduction by Ad vectors, as demonstrated by decreased serumlevels of AST at 3 dpi. We also demonstrated that wide-spreadpersistence of the modified vector genome in hepatic tissues at 28 and56 dpi actually represented only a fraction of the input vector genomespresent at 3 hours post infection (<14%) a result that has beendemonstrated by other groups after Ad vectors are allowed to persist invivo, ie: Ad mediated transduction of immune-incompetent animals.(Broughet al. (1997) J. Virol. 71:9206; Worgall et al., (1997) Hum. Gene Ther.8:37). Finally, each of these improvements were accomplished in adultanimals that were neither tolerized to the bacterial β-galactosidaseneoantigen, nor treated with potentially toxic agents thatnonspecifically blunted the immune system.

Several recent reports have suggested that the immunogenicity of thetransgene encoded by Ad vectors is primarily responsible for thetransience of Ad vectors in vivo (Chen et al., (1997) Proc. Natl. Acad.Sci USA 94:1645); Clemens et al., (1996) Gene Ther. 3:965; Morral etal., (1997) Hum. Gene Ther. 8:1275; Lusky et al., (1998) J. Virol.72:2022; Gao et al., (1996) J. Virol. 70:8934; Tripathy et al., (1996)Nature Med. 2:545). This view is substantiated by the observation thatboth null Ad vectors and Ad vectors encoding homologous transgenes canpersist in immunecompetent animals. (Morral et al., (1997) Hum. GeneTher. 8:1275; Tripathy et al., (1996) Nature Med. 2:545; Gao et al.,(1996) J. Virol. 70:8934). We have therefore specifically chosen toevaluate the ability of the [E1-,polymerase-] Ad vector to transduce thehighly immunogenic bacterial β-galactosidase transgene into C57BL/6mice. Several groups have previously demonstrated that Ad mediatedtransduction of the bacterial β-galactosidase gene results in a lack ofAd vector persistence (eliminated with one month) in a variety ofC57BL/6 tissues. (Morral et al., (1997) Hum. Gene Ther. 8:1275; Mcclaneet al., (1997) Pancreas 15:236; Michou et al., (1997) Gene Ther. 4:473;Dedieu et al., (1997) J. Virol. 71:4626; Gao et al., (1996) J. Virol.70:8934; Muhlhauser et al., (1996) Gene Ther. 3:145; Dematteo et al.,(1996) Gene Ther. 3:4). The demonstration that a significantly modified[E1-, polymerase-] Ad vector can overcome the barrier to neoantigenictransduction in this model challenges the hypothesis that the transgeneis the primary (or only) determinant of Ad vector persistence in vivo.The fact that other modes of gene transfer (such as AAV or direct DNAtransfer) can also allow for persistence of neoantigenic transgenes(including bacterial β-galactosidase) in immune-competent animalsfurther substantiates this view, independent of our findings.(Xiao etal., (1996) J. Virol. 70:8098; Wolff et al., (1992) Gene Ther. 1:363)

Recognizing that both vector and transgene encoded epitopes contributeto Ad vector elimination, we hypothesize that there may be two distinctimmune responses initiated after Ad mediated transduction, that we willrefer to as either “hit#1” or “hit#2”. Specifically, first-generation Advector-derived gene expression is responsible for “hit#1”, whileneoantigen expression (after Ad-mediated transduction) inimmunecompetent, nontolerant animals is responsible for “hit#2”. Thesimultaneous presence of both “hits” early after Ad vectoradministration results in a “two-hit” hyper-stimulation of the hostimmune response that results in the complete elimination of allAd-transduced cells, and in some instances may also break tolerance toself-antigens.(Tripathy et al., (1996) Nature Med. 2:545) The “two-hit”hypothesis can therefore accommodate our observation that modified Advectors can overcome the immunogenicity of transduced neoantigens, aswell as explain why null Ad vectors and Ad vectors encoding homologoustransgenes can also persist in immunecompetent animals, since in each ofthese situations only one “hit” of the “two hits” required to eliminateAd infected cells are elicited. The “two-hit” phenomenon may be due toseveral features unique to normal Ad biology, including the recentdemonstration that Ad vectors appear to efficiently infect antigenpresenting cells (APCs) while AAV based vectors do not (Jooss et al.,(1998) J. Virol. 72:4212). Finally, this hypothesis does not precludethe possibility that the Ad-vector (hit#1) stimulus may be potent enoughto provoke an immune-response to presumably homologous transgenes (insome instances), a situation analogous to the observation that Ad vectortransduction can sometimes even break tolerance to self-antigens.(Tripathy et al., (1996) Nature Med. 2:545).

EXAMPLE 17 Creation of IVa2 Vector

The C7 adenovirus packaging cell line was created with the use of aportion of the adenovirus genome which not only included the full lengthpolymerase coding region but also included a full length version of theIVa2 gene. In the introduced DNA, the IVa2 gene was also present in sucha manner that it was flanked by a potent CMV promoter/enhancer on its 5′end and a polyadenylation signal on the 3′ end (Amalfitano, et al.,(1997) Gene Therapy 4:258-263). Consequently, it was possible that thecell line might also be capable of expressing ample quantities of theIVa2 protein to allow transcomplementation of Ad IVa2 deletion mutants.We have determined that this is in fact the case, by creation of suchdeletion mutants and growing them to high titer on C7 cells. Thereforeit is now possible, according to the methods of the present invention,to create and propagate, without helper virus, Ad vectors containing aIVa2 deletion. Such vectors might also have deletions in E1, polymeraseand other deletions discussed herein and still be capable of growth tohigh titer on the C7 packaging cell line.

To create the Ad IVa2 deletion vector, a deletion was introduced into anadenovirus shuttling plasmid containing essentially nucleotides 0-358 ofthe adenovirus left end, an AscI site, followed by the adenovirussequences between map units 9 and 43 of the adenovirus genome. Theshuttling plasmid was then re-engineered by deleting out adenovirus DNAsequences that encompass not only the 3′ end of the polymerase gene, butalso a substantial portion of the IVa2 gene. This was achieved byrestriction enzyme digestion and subcdoning (effectively eliminating nt4830-5766 of the Ad5 genome, between Accl and Bst1107I sites) togenerate the final shuttling plasmid referred to a pAdAscLΔIV2, Δpol(FIG. 36). pAdAscLΔIV2, Δpol contains a 942 bp deletion at aboutnucleotides 4830 to 5766 and a second deletion of about 608 nucleotidesat about nucleotide 7274 to 7881 (as well as deleted E1 and E3 regions),effectively knocking out both IVa2 and polymerase function.

This shuttling plasmid was then co-transfected into the C7 packagingcell lines. Obviously the only way to generate such a virus would be ifthe packaging cell line produced enough IVa2 gene product totrans-complement the missing IVa2 gene activity in the resultant vector.We isolated such a virus, called [E1-, E3-, IVa2-, pol-]Ad anddemonstrated by restriction enzyme digestion that its genome is indeeddeleted for the IVa2 region. This virus is a unique vector demonstratingthe potential of the cell line to trans-complement any virus that isdeleted for the IVa2 regions of genes.

Other deleted adenoviruses carrying deletions in the IVa2 region as wellas other deletions can be generated using other shuttle vectors of thepresent invention by routine modifications of the techniques describedabove to generate the [E1-, E3-, IVa2-, pol-]Ad.

Shuttle plasmids pAdAscLΔIV2 (FIG. 19; deletion of about 942 bp at aboutnucleotides 4830 to 5766 of Ad5 genome in addition to deleted E1 and E3regions), pAdAscLΔIV2, Δpp (1.6) (FIG. 36; deletion of about 942 bp atabout nucleotides 4830 to 5766, a deletion of about 608 bp at aboutnucleotides 7274 to 7881, and a deletion of about 955 bp at aboutnucleotides 8631 to 9585 of the Ad5 genome in addition to deleted E1 andE3 regions), and pAdAscLΔIV2, Δpp (2.4) (FIG. 37; deletion of about 942bp at about nucleotides 4830 to 5766, a deletion of about 608 bp atabout nucleotides 7274 to 7881, and a deletion of about 2312 bp at aboutnucleotides 7274 to 9585 of the AdS genome in addition to deleted E1 andE3 regions) have been created. These shuttle plasmids are used to createthe corresponding deleted adenoviruses using any technique known in theart, for example, the method described above to generate [E1-, E3-,IVa2-, pol-]Ad. The vector [E1-, E3-, IVa2-]Ad is generated frompAdAscLΔIV2 (this vector also lacks polymerase function). [E1-, E3-,IVa2-, pol-, pTP-(1.6)]Ad and [E1-, E3-, IVa2-, pol-, pTP-(2.4)]Ad aregenerated from pAdAscLΔIV2, Δpp (1.6) and pAdAscLΔIV2, Δpp (1.6),respectively, using routine techniques known in the art or the methodsdisclosed herein.

It is now possible to increase carrying capacity of adenovirus vectorsby simple deletion of the entire IVa2 region of genes. This willsignificantly increase the versatility of Ad vectors be providing anincreased ability to carry larger gene constructs for potential genetherapy applications. Larger gene constructs are desirable sinceregulatory elements, specific enhancer promoter elements, and/or largergenes can only be accommodated by adenovirus vectors that havesufficient physical carrying capacity.

EXAMPLE 18 100K Deletion

In certain situations even additional transgene carrying capacity isdesirable. Further, deletion of genetic material coding for virusspecific proteins may further reduce the antigenic profile of thevectors and promote increased in vivo efficacy. One region in which suchdeletions proved to be possible is known as the 100K region of theadenovirus genome.

This region of the adenovirus genome encodes a protein known as 100K dueto its apparent molecular weight when observed in proteinelectrophoresis gels. The 100K protein encoded by the adenovirus acts asa scaffolding protein during final virion assembly in a host cell. The100K protein is not incorporated into the final virus particle. Finally,the 100K protein has a number of other functions which includestransport of other viral structural proteins from the cytoplasm into thenucleus of the host cell. This transport function includes the transportof the viral hexon protein. The hexon protein is a major structuralsubunit of the viral capsid. It has been demonstrated in past literaturethat lack of 100K activity results not only in lack of detectable 100Kprotein in infected cells but also destabilization of hexon monomerswhich results in their rapid degradation in the cytoplasm as well. It isimportant to recognize that that these previous observations wereobtained with the use of hexon (temperature sensitive) mutants. Theseare mutant viruses that have a single point mutation within thenucleotide sequence of the 100K protein which makes the mutant virusesonly viable at 32° C. and mutant at a temperature of 39° C.

The benefits of creating an adenovirus with the 100K gene deleted aremultiple. Deletion of the 100K gene increases carrying capacity ofresultant vectors that included this deletion significantly, (perhaps upto 10%) translating to approximately an additional 3000 base pairs ofcarrying capacity in an adenovirus vector. In addition, if 100Kdeletions can be included in adenovirus vectors that are deleted forother regions of the adenovirus genome as described elsewhere herein.The addition of these additional deletions significantly improves thevirus' in vivo gene transfer biological profile. This is because theresultant viral vector that is modified by multiple gene deletionsincluding deletion of 100K has a decreased ability to express multipleviral genes, including lack of expression of the hexon protein secondaryto hexon monomer destabilization due to lack of 100K activity. Finally,a vector deleted for 100K has a decreased potential to generatereplication competent adenovirus. This is because multiple recombinationevents would be required to regenerate a wild-type adenovirus. This isin contrast to conventional E1 deleted adenovirus vectors which onlyrequire a single recombination event to occur between the adenovirusvector and the resident E1 sequences present in human 293 cells togenerate a wild-type adenovirus.

A 100K expressing cell line was constructed by constructing a plasmidcontaining the 100K coding sequence of adenovirus subcloned between aCMV enhancer promoter element and a polyadenylation sequence to create a100K transgene cassette. The resulting plasmid pcDNA3+100K (pcDNA3obtained from Invitrogen Corp.) was linearized by restriction digestionand transfected into human 293 cells using the calcium phosphatetransfection technique. The pcDNA3+100K plasmid not only encoded the100K transgene cassette but also a transgene cassette encoding G-418Rresistance. Therefore after transfection cells were exposed to thecytocidal agent G418 and cell clones that were resistant to G418 weresubcloned and expanded. These subcdones represented cells that hadintegrated the 100K expressing plasmid into their genome, and were alsoexpressing high levels of the G418 resistance transgene. Δpproximately20-25 clones that were G418 resistant were subcloned and propagated.

Subsequently each clone was exposed to a virus known as H5ts116(obtained from the laboratory of Dr. Ginsburg at the ColumbiaUniversity). This virus contains a mutation in the 100K gene region thatis temperature sensitive. That is the virus only grows to high levels ata temperature of 32° but not at 39°. The 100K temperature sensitivemutant virus was used to screen each of the G-418 resistant cell linesas follows. Each of the cell lines was infected with the H5ts116 virusat the non-permissive temperature of 39° C. Theoretically, any cell linewhich could produce high levels of the 100K protein would allow growthof the mutant virus even at the non-permissive temperature of 39° C. OneG418 resistant cell clone referred to as K-16 was found to allow growthof the virus H5ts116 at the non-permissive temperature of 39° C.

This cell line, K-16, was expanded and investigated more thoroughly. TheK-16 cell line had integrated into its genome the 100K sequencesconfirming that it indeed was capable of expressing a functional 100Kprotein. In addition, K-16 cells were producing very high amounts of100K protein specific RNA molecules. This again demonstrated that thecell lines were producing very high levels of 100K and confirmed thecells were capable of growing the virus H5ts116 at non-permissivetemperature (FIG. 20). FIG. 20 shows the results of PCR amplification ofgenomic K-16 DNA using 100K specific primers (Panel A). Equivalentamounts of mRNA from the 293 cell line and the K-16 line were originallyelectrophoresed and transferred (FIG. 20, Panel B). But as shown byPanel C, only the K-16 cell derived mRNA contained sequences specificfor 100K. Panel C shows the presence of the mRNA band in K-16 afterprobing with a 32-P labeled DNA fragment specific for 100K.

Finally, Table I, below, shows that only the K-16 cell line permitscytopathic growth at the restrictive temperature of adenovirus H5ts116which contains a temperature sensitive mutation in the gene coding forthe 100K protein. In addition, since K-16 was derived from human 293cells which constitutively produce adenovirus E1 gene products, we haveconfirmed that the co-expression of the E1 genes and the 100K genes isnot cell toxic. This latter fact is advantageous because it allowsproduction according to the methods of the present invention, of avector that is simultaneously deleted not only for the adenovirus E1genes but also for the adenovirus 100K genes.

TABLE I Growth of H5ts116 on K-16 Cells and 293 Cells H5ts116 infectionH5ts116 infection @ @ Cell Type 32.0° C. 39.0° C. 293 Cells (E1+) +++ 0K-16 Cells (E1+, +++ +++ 100K+) Key: 0 = no cytopathic effect; +++ = allcells undergo cytopathic effect

In addition, we have also introduced the 100K expression plasmid intothe C7 packaging cell line which already expresses the E1 gene productas well as the adenovirus polymerase and preterminal protein genes. Thisadenovirus packaging cell line can simultaneously transcomplementvectors deleted for the E1 polymerase, preterminal protein, IVa2, and100K gene regions.

We have demonstrated via Northern Blot analyses that we can achieve highlevel expression of the 100K gene in C7 cells. FIG. 21 shows 32-Plabeled 100K DNA probes of cell mRNA from four isolates of C7 cells intowhich the 100K plasmid was introduced. Each lane represents anindividual cell line. Total RNA from each cell line was electrophoresed,blotted and probed with 32-P labeled 100K specific DNA. C7 cellsexpressing the 100K gene can be used to produce vectors with acombination of E1, Δpol, ΔpTP, IVa2 and/or 100K deletions. Vectorsdeleted for all of these regions may have improved characteristics forutilization in gene therapy applications since the viruses would havesubstantially increased carrying capacity, a decreased ability togenerate replication competent adenovirus, and finally, an improved invivo biological profile since they would lack the ability to expressmultiple viral gene products.

EXAMPLE 19 In vivo Administration of Ad Δpol Vectors

Ad Δpol Vector Administration: The construction of the modifiedAdLacZΔpol vector (deleted for both E1 and polymerase activities) aswell as Adsub360LacZ, and their high-titer production was as describedin Example 1 and 2. Six-8 week old immune-competent C57BU6 mice (JacksonLaboratory, Bar Harbor, Me.) were intravenously (iv) injected with 200μl of a PBS solution containing 4×10⁹ β-galactosidase forming units(BFU) of either AdLacZsub360 or AdLacZΔpol and subsequently sacrificedat the indicated time points. At least three animals were analyzed pertime point, except animals administered Adsub360LacZ at 56days-post-infection (dpi), where n=2. All animal procedures andsubsequent analyses were performed as approved by the Duke UniversityInstitutional Animal Care and Use Committee. Liver and plasma samplesharvested from each of the animals were processed as described below.

In-situ Bacterial β-Galactosidase Determination: Liver specimens wereembedded in OCT compound, and snap-frozen in liquid-nitrogen cooledisopentane. 10 μm frozen sections were obtained with a Leica cryostatand briefly cross-linked in a 0.5% glutaraldehyde/PBS solution for 5min. The fixed sections were then placed into 1 mM MgCl₂ PBS for 20min., and incubated overnight at 37.0° C. in a solution containing 5 mMpotassium ferricyanide, 5 mM potassium ferrocyanide, 1 mM MgCl₂ and 1mg/ml of the chromogenic X-gal substrate. The stained sections werecounterstained with eosin, dehydrated, mounted in Permount, andphotographed.

Bacterial β-Galactosidase Activity Determinations: Total protein wasextracted from liver tissues by freezing in liquid nitrogen, followed byhomogenization in lysis buffer (100 mM potassium phosphate pH 7.8, 0.2%Triton X-100, 0.5 mM DTT) and centrifugation at 14,000 RPM. The proteinconcentrations in the liver extracts was determined via the BCA proteinassay kit (Pierce: Rockford, Ill.). Twenty-five micrograms of proteinfrom each sample was incubated at 37° C. for 1 hour with 1 mM MgCl₂, 45mM β-mercaptoethanol, 0.264 μg of o-nitrophenyl-β-D-galactopyranoside(ONPG), and 50 mM sodium phosphate solution at pH 7.5. The reaction wasstopped with 0.5M NaCO₃, and the absorbance of each sample wasdetermined at a wavelength of 420 nm. Bacterial p-galactosidasestandards (Sigma, St. Louis, Mo.) diluted in lysis buffer were used togenerate a standard curve, and the β-galactosidase levels detected ineach extract was determined by comparison with the standard curves.

RNA Isolation and Analysis: Mouse liver total RNA was extractedutilizing the RNA Isolation Kit R-5500 (PGC Scientific, Frederick, Md.)as per the manufacturer's specifications. Δpproximately twentymicrograms of each RNA sample was separated in a 0.8%formaldehyde-agarose gel, transferred to a nylon membrane, and probedwith a [α-³²P] dCTP -labeled DNA probe capable of hybridizing tobacterial β-galactosidase mRNA transcripts derived from either vector.The probed membranes were washed, and exposed to radiographic film.Densitometric analysis of all exposed films was done with the NIH-Imagesoftware package. Quantitative comparison of expression levels wasachieved by comparing the ratios of LacZ mRNA detected, to the total RNAloaded (as determined by 18s rRNA quantitation) for each sample.

DNA Isolation and Analysis: DNA was extracted as previously described,with some modifications (Amalfitano et al., (1996) Muscle Nerve19:1549). Briefly, 100 mg of minced mouse liver tissue was mixed with600 μl of TNES buffer (10mM Tris-Cl pH 7.5, 400 mM NaCl, 100 mM EDTA and0.6% SDS) and 17.5 μl of proteinase K (20 mg/ml). After overnightincubation at 37° C., 167 μl of 5M NaCl was added and the samples werecentrifuged at 14,000 RPM for 10 min. The supernatant was treated withRNase A (5 μg/ml) and extracted with phenol-chloroform. Total DNA wasprecipitated with 95% ethanol, washed in 70% ethanol, air dried andresuspended in TE buffer. Twenty micrograms of total liver DNA from eachmouse were digested with EcoRI, electrophoretically separated in a 0.7%agarose gel, and transferred onto a nylon membrane. Control samples of20 μg of liver DNA extracted from non-infected animals spiked witheither 1 or 10 copies of AdLacZΔpol virus genomes/ hepatocyte genomewere also included as internal standards. The membrane was crosslinked,and hybridized to a [α-³²P] dCTP labeled DNA probe (˜6 kb EcoRIsubfragment of AdLacZΔpol) capable of detecting either vector. Themembrane was washed and subsequently exposed to autoradiography films aspreviously described (Amalfitano et al., (1998) J. Virol. 72:926).

Histopathological Studies: Mouse liver tissues were fixed in 10% neutralformalin and embedded in paraffin. 5 μm thick paraffin sections werestained in hematoxylin-eosin, observed under microscope, andrepresentative sections were photographed at equivalent magnifications.

Detection of Aspartate Aminotransferase (AST): Blood samples from eachof the infected mice were harvested via retro-orbital bleeding, andplasma isolated after brief centrifugation. Plasma AST levels weremeasured in duplicate by using Sigma® AST diagnostic kit 505 accordingto the manufacturer's specifications. Plasma samples collected from 6age-matched, uninfected C57BL/6 mice were also analyzed and depicted.

EXAMPLE 20 Persistence of Bacterial β-Galactosidase Activity afterHepatic Transduction of Immunocompetent, Nontolerant Mice

As described in Example 1, we have isolated a packaging cell line thatsupports the high level growth of a modified Ad vector(“AdLacZΔpol”-deleted for both E1 and polymerase activities) thatencodes the highly immunogenic transgene, bacterial β-galactosidase. Theability of AdLacZΔpol to allow for sustained transduction of bacterialβ-galactosidase derived enzyme activity was compared with that of afirst-generation, [E1-] Ad vector. C57BL/6 mice are an immune-competentstrain of mouse that has been repeatedly demonstrated to be non-tolerantof the bacterial β-galactosidase transgene (see, e.g., Morral et al.,(1997) Hum. Gene Ther. 8:1275; McClane et al., (1997) Pancreas 15:236;Michou et al., (1997) Gene Ther. 4:473). After the intravenousadministration of 4×10⁹ β-galactosidase forming units (BFU) ofAdsub360LacZ (E1 deleted) transduction of nearly 100% of the liverhepatocytes was achieved, as visualized by widespread staining with theX-gal chromogen (FIG. 22, Panel A). Dramatically, by 28 dayspost-infection (dpi) no X-gal staining cells remained (FIG. 22, PanelC). As expected, the same result was noted at 56 dpi (FIG. 22, Panel G).These results were in agreement with previous reports demonstrating thattransduction of C57BL/6 mouse hepatocytes with [E1-] Ad vectors encodingneoantigens are rapidly eliminated within one month of initialadministration (see, e.g., Morral et al., (1997) Hum. Gene Ther. 8:1275;McClane et al., (1997) Pancreas 15:236; Michou et al., (1997) Gene Ther.4:473).

In contrast, an identical infection of C57BL/6 mice with the AdLacZΔpolvector (E1 and polymerase deleted) resulted in high level andwide-spread expression of bacterial β-galactosidase both at 3 dpi (FIG.22, Panel B) as well as at 28 dpi (FIG. 22, Panels D-F). Each of thelatter panels represented individual test animals. Review of multiplesections demonstrated that 75-100% of the hepatocytes were still X-galstained at 28 dpi, indeed the only cells not stained in the sectionswere periportal inflammatory cells (see below). This result demonstratedthat utilization of the modified Ad vector allowed for an avoidance ofthe immune responses that normally eliminate most of the cellstransduced by Ad vectors encoding neoantigens in immunecompetentanimals. However, X-gal staining of AdLacZΔpol transduced livers at 56dpi demonstrated a lack of X-gal staining in all liver sections in eachof the treated groups of animals (FIG. 22, Panel H).

The X-gal staining data was also quantified by direct measurement ofbacterial β-galactosidase activity in liver-derived protein extractsfrom each of the experimental animal sets (FIG. 23). Again, at 28 dpisubstantial amounts of bacterial β-galactosidase activity were detectedonly with the use of AdLacZΔpol vector. Absolute levels of bacterialβ-galactosidase activity had significantly decreased (˜92% drop) between3 and 28 dpi in the AdLacZΔpol treated animals, a decrease that directlycorrelated with reduced amounts of bacterial β-galactosidase mRNAexpression (see below). Furthermore, a lack of bacterial β-galactosidaseactivity in liver tissues from 56 dpi animals confirmed the previousin-situ results. The lack of expression of bacterial β-galactosidaseactivity at 56 dpi in AdLacZΔpol treated animals was therefore exploredfurther.

EXAMPLE 21 Lack of Transgene Activity at 56 dpi is not Due to a Lack ofModified Vector-Genome Persistence

Total RNA extracted from liver sections derived from each of theexperimental animals was isolated and analyzed with a bacterialβ-galactosidase specific probe (FIGS. 24 and 25). In each animal, lackof X-gal staining correlated with a lack of detectable bacterialβ-galactosidase mRNA expression. Specifically, by 28 dpi only AdLacZΔpoltreated animals had significant levels of bacterial β-galactosidasespecific RNA remaining. Though present, the average amount of bacterialβ-galactosidase specific RNA in AdLacZΔpol treated animals at 28 dpi wasapproximately 86% of the levels noted at 3 dpi, a decrease thatcorrelated with the approximately 92% drop in enzyme activity noted at28 dpi (FIG. 23). Notably, at 56 dpi AdLacZΔpol treated animals had acomplete absence of bacterial β-galactosidase specific RNA, a resultthat correlated with a complete lack of enzyme activity.

To determine whether the lack of transgene specific RNA expression at 56dpi was due to a lack of AdLacZΔpol genome persistence, total DNA wasextracted from each of the experimental animals and probed with an Adspecific probe (FIG. 26). As expected, Adsub360LacZ DNA was notdetectable at 28 or 56 dpi. However, we were readily able to detectsignificant amounts of vector specific-DNA sequences in AdLacZΔpoltreated animals both at 28 and 56 dpi. Since up to 100% of theAdLacZΔpol transduced hepatocytes were X-gal stained at 28 dpi, theamount of vector DNA detected at 28 and 56 dpi was a reflection ofpersistent and widespread hepatic transduction. Consequently the rapidloss of X-gal staining, bacterial β-galactosidase enzyme activity, andbacterial β-galactosidase mRNA expression at 56 dpi was not due to alack of vector persistence, but was rather due to a lack of transgenederived RNA expression from the persistent vector genomes. Quantitationby densitometry demonstrated that the absolute levels of vector DNAdetected both at 28 and 56 dpi averaged ˜4.4 copies of the AdLacZΔpolgenome per hepatocyte, a result consistent with the widespread X-galstaining data presented in FIG. 22, Panels D-F. Interestingly, this DNArepresented at most 14% of the input vector DNA noted at 3 hourspost-infection (data not shown), a result consistent with previousobservations in immune-incompetent mice. The latter studies havedemonstrated that a significant amount of input Ad vector genomes arecleared by innate mechanisms present in the liver within 24 hours ofinfection, independent of immune responses directed to the vector ortransgene. (Brough et al., (1997) J. Virol. 71:9206; Worgall et al.,(1997) Hum. Gene Ther. 8:37).

EXAMPLE 22 Inflammatory Responses to Modified Ad Vectors

Histopathological evaluation demonstrated that a significantcellular-inflammatory infiltrate was evident in both sets of treatedanimals by 28 and 56 dpi (FIG. 27). Since both vectors elicit similarreactions at 28 and 56 dpi, the inflammatory infiltrates may have beendirected to stimuli common to both vector preparations. The infiltratescontained lymphocytes, macrophages, and occasional plasma cells. Incontrast, at 3 dpi, levels of serum AST (released by damagedhepatocytes) were significantly less (p=0.01) in AdLacZΔpol treatedanimals than in animals infected with the first-generation Ad vector(FIG. 28). After 28 dpi, however, both experimental animal sets returnedto base line serum AST activities. It was significant that at 3 dpi, thedecreased serum AST levels noted after AdLacZΔpol hepatic transductionpositively correlated with the lack of elimination of AdLacZΔpoltransduced hepatocytes noted at 28 and 56 dpi.

EXAMPLE 23 Isolation of the GAA Transgene

The acid α-glucosidase full length cDNA (Hoefsloot, L. H. et al. (1988)EMBO Journal 7, 1697-1704), was excised with EcoRI from the pSHAG2vector (Hoefsloot, L. H. et al. (1990) Biochem. J. 272, 485-492),isolated on an agarose gel, and ligated into the multicloning site ofthe widely used and commercially available mammalian expression vectorpcDNA3 (Invitrogen, San Diego, Calif.). Because EcoRi is used as acloning site there are two possible ligation products, with oppositeorientations. The plasmid which has the GAA transcription in thedirection under the control of the CMV promoter was selected byrestriction enzyme digestion according to the predicted map. Theresultant plasmid was called pcDNA3-GAA and contains the CMV promoter.

The CDNA was also cloned as a polycistronic construct in the EcoRIrestriction site of the pMT2 vector (Kaufman, R. J., (1990) MethodsEnzymol. 185, 537-566; Kaufman, R. J., (1990) Methods Enzymol. 185,487-511), and called pMT2-GAA. A 1714 base pair fragment containing theDHFR gene under the control of the adenovirus major late promoter, theTPL (adenovirus tripartite leader) the intervening sequence (IVS) andthe SV40 poly A site, but not the SV40 enhancer, was excised by BamHI,and isolated on an agarose gel. This fragment was ligated into thecompatible BgIII restriction site of the pcDNA3-GAA plasmid. In thisselected plasmid, called pJW55, the orientation of the transcription ofGAA and of DHFR is the same.

A 567 bp fragment of the GAA cDNA, starting from 18 bp 5′ of the ATGstart codon and ending 529 bp into the coding sequence of acidα-glucosidase, was prepared by PCR amplification using the senseoligonucleotide primer GA224(+) TCC AGG CCA TCT CCA ACC AT (SEQ ID NO:1)and the antisense oligonucleotide primer GAA751(−) TCT CAG TCT CCA TCATCA TCA CG (SEQ ID NO:2). This fragment contains the natural Sacil siteof the GAA cDNA which site occurs 320 bp into the acid α-glucosidasecoding sequence. The 567 bp fragment was ligated into the pCRII plasmidusing the TA cloning technique and following the instructions of thesupplier (Invitrogen, San Diego, Calif.). This results in two possiblecloning products with opposite orientations. We chose the one that hasthe GA224(+) site close to the HindIII site of the original PCRIIplasmid. This plasmid is called pCRII-1. The orientation and the DNAsequences were confirmed by DNA sequencing and restriction enzyme digestmapping. pCRII-1 contained the HindIII site 5′ relative to the ATG startcodon as well as the natural SacII site at 320 bp into the codingsequence of acid α-glucosidase.

Following digestion of pCRII-1 with both HindIII and SacII, a fragmentcontaining nucleotides 892-1496 is isolated on agarose gel. Thisfragment contains the nucleotides 279-670 of the original GAA cDNA. TheASCII site is 314 nucleotides downstream of the ATG start site. TheHindIII site is derived from the multiple cloning site of the originalplasmid vectors pCRII and pcDNA3.

The pcDNA3-GAA vector is similarly digested with both HindIII and ASCII,and the large plasmid fragment isolated on agarose gel. The smallfragment contains the beginning of the acid α-glucosidase gene,including the entire untranslated sequence prior to the ATG start codonand extending through the 3′ end of the SacII cleavage site, which is bp670 of the gene sequence. This small fragment is discarded. The largefragment contains that portion of the acid α-glucosidase gene sequencebeginning on the 5′ end of the SacII cleavage site at bp 671 andextending through the final bp of the gene which remains attached to theplasmid backbone.

The fragment containing nucleotides 279 through 670 of the original GAAcDNA isolated from the pCRII-1 digestion is now ligated into the largeplasmid fragment isolated from the pcDNA3-GAA digestion, creating theplasmid vector pcDNA3-5′sGM, which contains the 5′ shortened version ofGAA.

A fragment (nucleotides 898A4439) containing this newly createdshortened cDNA is excised from pcDNA3-5′sGAA by digestion with therestriction enzymes KpnI and XhoI. In pJW55 the full-length GAA cDNAinsert is removed by digestion with KpnI and Xho1 (nucleotides2611-6355), and the plasmid backbone isolated on agarose gel. Theshortened cDNA isolated from pcDNA3-5′sGAA is then ligated into thecompatible Kpn1-Xho1 sites of this plasmid backbone creating pJW-5′sGAA.Both Kpn1 and Xho1 sites are derived from the original pcDNA3 plasmidmultiple cloning site and are unique in both pJW55 and pcDNA3-5′sGAA.The resulting pJW-5′sGM plasmid has the 5′ shortened non-ranslatedsequence of GAA under the CMV promoter as well as the DHFR gene underthe adenovirus Major Late promoter. The cDNA insert starts at nucleotide2611 and ends at nucleotide 6153.

EXAMPLE 24 Creation of Ad Vector with hGAA Transgene

Plasmid pcDNA3GAA was digested by XmnI, and the approximately 4.5kbfragment containing the transgene cassette was blunt ended with T4 DNApolymerase and subdloned into the shuttling plasmid of the presentinvention, pAdAscLΔpol (Example 2) which is designed for theconstruction of Ad vectors deleted for the E1, E3 and polymerase genefunctions. The resulting plasmid pAdAscLΔpolhGM was co-transfected viathe calcium phosphate technique (Jones et al. (1978) Cell 13:181;Mittereder et al., (1996) J. Virol. 70:7498) into the adenoviruspackaging cell line C-7, simultaneously with XbaI, Clal and ScaI digestsof adenovirus virion DNA from dl7001. Recombinant vector clones wereexpanded and confirmed to contain the hGAA gene by restriction enzymedigestion of vector derived DNA (Jones et al. (1978) Cell 13:181), aswell as by multiple functional analyses (see Examples below). Cesiumchloride purified AdhGMΔpol vector was produced after infection (MOI was˜10) of 60, 150 mm tissue culture plates containing C-7 cells(expressing the adenovirus E1, polymerase, and preterminal protein). Allvector titers were confirmed by plaque forming unit (PFU) assay ofserial dilutions of the vector preparations of C-7 cells.

EXAMPLE 25 In vivo Administration of AdhGAAΔpol

Intravenous Administration: 1×10⁹ pfu of AdhGAAΔpol vector wasintravenously administered (via the retro-orbital sinus) into6.5-month-old C57BL/6 (wild-type) mice or 2-month-old GAA-KO mice(6^(neo)/6^(neo)). (Clemens et al., (1996) Gene Ther. 3:965). At therespective time points post-injection, plasma or tissue samples wereobtained and processed as described below. All animal procedures weredone in accordance with the Duke University Animal Care and UseCommittee guidelines.

Measurement of Tissue/Plasma GAA Activity and Glycogen Accumulation:Tissues were snap frozen in liquid nitrogen, homogenized in water, andinsoluble proteins were removed by centrifugation. The protein contentof the resultant lysates was quantified by the Bradford assay. Forplasma GAA activity detection, blood samples were collected byretro-orbital sampling into heparinized capillary tubes, followed byplasma isolation. GAA activity in each of the tissues or plasma wasassessed by measurement of 4-methyl-umbelliferyl-α-D-glucoside cleavageat pH 4.3, as previously described (Amalfitano et al., (1997) Gene Ther.4:258). Glycogen content was determined by treatment of tissue extractswith A. niger amyloglucosidase and measurement of glucose released. Allextracts were also assessed for background glucose release, i.e., in theabsence of the A. niger amyloglucosidase. Final glycogen content valueswere then determined after untreated glucose levels were subtracted fromthe glucose content of each of the amyloglucosidase treated tissueextracts (n=4 for C57BI/6 mice, n=3 for GM-KO mice, n=3 for Ad treatedGAA-KO mice).

Morphological Assessment of Tissues: Sections were placed in embeddingcompound and snap frozen in liquid nitrogen-cooled isopentane. Tenmicron sections of the tissues were collected with a Leicacryomicrotome, PAS stained, and visualized with a Leica microscope anddigital camera.

RNA Analysis: Total RNA was isolated from portions of mouse tissuesafter homogenization in 4.0 M guanidinium thiocyanate and cesiumchloride purification. 12.5 μg of total RNA from each tissue waselectrophoretically separated in a 1% formaldehyde-agarose gel, blottedonto a nylon membrane, UV crosslinked, and probed with a ³²P-labelled3.3 kb fragment containing the hGM cDNA. The probed membranes werewashed, exposed to autoradiographic film, and photographed.

Immunoblot Detection of hGAA: Respective mouse tissues were frozen,homogenized, centrifuged to remove insoluble proteins, and proteincontent of the supernatants was measured by the Bradford assay.Equivalent amounts of protein were electrophoretically separated in a10% polyacrylamide-SDS gel, transferred to a nylon membrane, and probedwith a rabbit anti-human GAA polyclonal antibody. Detection of the boundanti-hGAA antibody was visualized with the ECL detection system(Amersham). For direct detection of hGAA precursor in plasma, 2.5 μL ofeach sample was electrophoretically separated, followed by anti-hGAAantibody probing of the immunoblot as described above.

EXAMPLE 26 Construction of the AdhGAAΔpol Vector-Results

The production and clonal purification of modified Ad vectors deletedfor both the Ad E1 and polymerase genes has previously been described(Jones et al. (1978) Cell 13:181). These vectors have the ability toallow for prolonged persistence after hepatocyte infection, despiteneoantigen gene transfer (Ilan et al., (1997) Proc. Natl. Acad. Sci. USA94:2587). The construction of the AdhGAAΔpol vector was carried out asdescribed in Example 24, and is depicted in FIG. 29. Restriction enzymemapping of DNA derived from the purified vector confirmed that itcontained the CMV-hGAA transgene cassette (data not shown). Furtheranalysis demonstrated that the AdhGAAΔpol vector was capable of highlevel expression of the hGAA enzyme activity (as assessed by cleavage of4-methyl-umbelliferyl-α-D glucoside) after infection of cultured human293 cells (FIG. 30). Importantly, the supernatant overlying the infectedhuman cells accumulated increasing amounts of hGAA activity with time,suggesting that AdhGAΔpol infection resulted in both high levelexpression and sustained secretion of an active hGAA enzyme.

EXAMPLE 27 Hepatic Targeting of the AdhGAAΔpol Vector and GAA Secretion

Ad vectors preferentially transduce hepatocytes after intravenousadministration. The hypothesized model for GSD-II treatment thatcapitalizes upon hepatic targeting of Ad vectors was investigated asfollows: High level production and secretion of hGAA in the plasma wasdemonstrated after the intravenous injection of 1×10⁹ plaque formingunits (pfu) of AdhGMΔpol into wild-type mice (FIG. 31). Extremely highlevels of GAA activity were detected in the plasma as early as two daysafter infection, although the levels diminished with time. Inhepatocytes, a rapid down-regulation of CMV enhancer activity occurs,and may have contributed to the decreased GAA activities detected in theplasma, although production of murine antibodies to the human enzyme mayhave also been present. (Ilan et al., (1997) Proc. Natl. Acad. Sci. USA94:2587; Kaplan et al., (1997) Hum. Gene Ther. 8:45; Kay et al., (1995)Nat. Genet. 11:191).

Intravenous injections of the AdhGAAΔpol vector into the GAA-KO mousewere also done to ascertain the effectiveness of the secreted hGAA toreduce glycogen accumulations in affected skeletal and cardiac muscle.The GAA-KO mouse was previously generated by targeted knockout of themurine GAA gene and has a phenotype that includes systemic glycogenaccumulations in the cardiac and skeletal muscles, as well asprogressive clinical myopathy. (Clemes et al., (1996) Gene Ther. 3:965).After intravenous injection of the AdhGAAΔpol vector, high levels ofenzyme activity were demonstrated in the plasma of the GAA-KO mice (FIG.32). Importantly, protein immunoblot analysis of plasma demonstratedthat the predominant form of hGAA detected in the treated animals at 3days post infection (dpi) had a molecular weight of ˜110 kD, a sizeequivalent to the precursor (unprocessed) form of hGAA (FIG. 32)(Kochanek et al., (1996) Proc. Natl. acad. Sci. USA 93:5731). Inaddition, a significant amount of the processed from of the enzyme (˜77kD) was also detected in the plasma. This may be the result ofproteolysis of the mature GAA in the plasma, or release of the matureform of hGAA from infected hepatocytes.

EXAMPLE 28 Assessment of hGAA Distribution in the GAA-KO Mouse

After treatment with the AdhGAAΔpol vector, GAA-KO animals weresacrificed and tissues were analyzed for the presence of GAA activity. Apilot study demonstrated that at 4 dpi GAA activity was detected inmultiple muscle tissues of AdhGAΔpol treated GAA-KO mice, but glycogenlevels had only minimally begun to decrease (data not shown). By 12 dpiGAA activity levels in the quadriceps, gastrocnemius, diaphragm, andcardiac muscles of the Ad treated GAA-KO animals were all significantlyelevated when compared to the enzyme levels detected in untreated GAA-KOmice (FIG. 33). Even more dramatically, the muscle GAA enzyme activitylevels detected in the treated animal exceeded those detected inwild-type mice (FIG. 33) to confirm that the GAA activity detected inthe muscles of treated animals was not due to de novo biosynthesis ofGAA in the muscles, RNAs derived from various tissues were also analyzed(FIG. 34). Despite the fact that the heart and quadriceps muscles ofAdhGAΔpol treated GAA-KO mice contained high levels of GAA activity, GAAspecific RNA transcripts were detected only in the liver, and not ineither of the muscle tissues. Note that in a wild-type mouse, GAA mRNAwas readily detected in muscle tissues, despite the fact that the enzymeactivities in these tissues were lower than in the AdhGAAΔpol treatedanimal. Immunoblot analysis of protein extracts derived from the liver,cardiac, and quadriceps muscles of the Ad treated GAA-KO mice alsodemonstrated that the liver had high levels of the precursor form ofhGAA present, while the cardiac and quadriceps muscles did not (data notshown). Based upon these observations, we concluded that the enzymeactivities detected in the muscle tissues of the AdhGAΔpol treatedanimals was most likely derived exogenously, ie., via uptake ofprecursor GAA secreted by the liver.

EXAMPLE 29 Systemic Reversal of Muscle Glycogen Accumulation in GAA-KOMice after AdhGAAΔpol

This study was undertaken to confirm that the enzyme activities detectedin the muscles of the Ad treated GAA-KO mouse resulted in correctlysosomal targeting of the enzyme. Previous studies have demonstratedthat exogenously administered precursor GAA can target to lysosomes andact to reduce intra-lysosomal glycogen accumulations in both the AMDquail, and the GAA-KO mouse (Amalfitano et al., (1997) Gene Ther. 4:258;Armentano et al., (1997) J. Virol. 71:2408). Correct lysosomal targetingof the hepatocyte secreted hGAA should also result in a reduction ofglycogen accumulation in a number of muscles in the AdhGMΔpol treatedGAA-KO mice. In situ periodic acid-Schiff (PAD) staining for glycogenaccumulation in multiple muscles of untreated and AdhGAAΔpol vectortreated GAA-KO mice were evaluated (FIG. 35). In each of the respectivemuscle tissues both granular and diffuse forms of glycogen accumulation(these staining patterns represented accumulation of glycogen inlysosomes and cytoplasm, respectively) were significantly reduced in theAdhGAAΔpol vector treated GAA-KO mice, in comparison to the stainingobserved in untreated GAA-KO mice. This result was also confirmed afterquantification of total intra-cellular glycogen levels in a variety oftissues derived from the treated mice (Table II). The heart anddiaphragm muscles appeared to be especially responsive to the AdhGAAΔpoltreatment, an important observation since cardiac and/or respiratorymuscle involvement are the primary causes of mortality in the variousforms of GSD-II. Also of note, exogenous administration of a breastmile-derived form of hGAA did not significantly correct the glycogenaccumulations noted in the muscles of GAA-KO mice until 6 months afterinitiation of therapy, suggesting that hepatically secreted hGAA may bea more potent form of the enzyme (Cox et al., (1993) Nature 364:725).These results confirmed that the increased GAA activities noted in themultiple muscles of AdhGAAΔpol vector treated GAA-KO mice resulted inlysosomal targeting of the enzyme, and phenotypic correction of theprimary defect in GSD-II, namely significant reductions in muscle cellglycogen accumulation.

It was further observed that in GAA knockout mice less than 2% ofwild-type levels of GAA are present in a variety of muscle tissuesanalyzed, facilitation detection of the exogenously produced hGAA. Highlevels of hGAA activity were again noted in the serum of infected GAA-KOanimals after intravenous administration. Levels similar to that notedafter infection of wild type mice. Furthermore, the isoform of theenzyme detected in the serum consisted of substantial amounts of theunprocessed 110 kD precursor form of hGAA, in addition to the processed76 kD isoform, based upon immunoblotting results (FIG. 32). Importantly,it is the precursor form of GAA that is predicted to be amenable tomannose-6-phosphate uptake mechanisms present in both skeletal andcardiac muscle cells. In further substantiation of this data, eventhough GAA activities were significantly diminished (less than 90% ofwild-type levels) in a variety of tissues in the GAA knockout animals,those animals receiving the AdhGAΔpol vector of the invention hadextremely high levels of GAA activity (exceeding the activities detectedin wild-type animals) in all tissues analyzed (FIG. 33). These resultsdemonstrate that after a single, intravenous administration of amodified Ad vector allowing for the extended expression of hGAA,systemic distribution of hGAA to the muscle tissues primarily affectedin Pompe's disease can be achieved.

The presence of significant amounts of the precursor hGAA enzyme in theserum of AdhGAA treated animals results in what we believe to bereceptor mediated uptake (mannose-6-phosphate or other) andintra-cellular, lysosomal targeting of hGAA. Quantitative measurementsof glycogen the content of the tissues as determined by enzyme activityassay confirmed that the treated animals showed dramatically reducedglycogen accumulation 12 days after treatment (FIG. 35). It is importantto note that despite decreased presence of precursor enzyme in the serumof treated animals at 12 dpi (FIG. 32) glycogen content of the tissueswas still significantly reduced, concomitant with persistent GAA enzymeactivity in the same tissues at 12 dpi. Therefore this data confirmedthat intravenous administration of a modified Ad vector can allow forsustained levels of lysosomal enzymes to be released into the systemiccirculation, with subsequent uptake into target tissues where the enzymecan act to correct tissue pathology.

These results demonstrate that the liver can serve as an exocrine gland,that adenovirus vectors of the present invention can infect up to 100%of liver cells after a single intravenous administration, and thatsufficient quantities of hGAA are excreted by the infected liver cellsto cause dramatically increased enzyme levels of both 76 kD and 110 kDenzyme isoforms in the animal's serum and, finally, that the enzyme isreadily taken up by skeletal and cardiac muscle cells of the animalresulting in a dramatic reduction in glycogen accumulations.

TABLE II Glycogen Content (μmole/mg) GAA-KO + Tissue Wild-type GAA-KOAdhGAAΔpol Heart .0078 ± .0130 2.4100 ± .9200  .0032 ± .0045 Diaphragm.0390 ± .0350 .9300 ± .3300 .1100 ± .1600 Quadriceps 0 ± 0 .8800 ± .1200.1290 ± .0400 Gastrocnemius .0365 ± .0260 .9300 ± .0700 .1170 ± .1380Liver .4600 ± 3800 .2300 ± .0170 .0206 ± .0200

EXAMPLE 30 Production of Deleted Adenovirus Vectors via BacterialHomologous Recombination

Here we describe another strategy to rapidly generate multiply deletedrecombinant adenoviruses. This strategy approach uses the plasmidpAdEasy1 (an Ad5 viral backbone that is E1 and E3 deleted with abacterial origin of replication) and a second shuttle plasmid thatcontains homologous regions to the pAdEasy plasmid as initiallydescribed by He et al, (1998) Proc. Natl. Acad. Sci. USA 95:2509; U.S.Pat. No. 5,922,576. The homologous regions in the shuttle plasmid flanka multiple cloning site where subcloned transgenes are ligated. Whenthese two plasmids are co-introduced into bacteria (bacteria that allowfor increased frequencies of homologous recombination between DNAmolecules generally) recombination between the homologous regions of theplasmids results in the generation of a full length adenovirus vectorgenome, but as a bacterial plasmid containing a bacterial origin ofreplication and a selectable marker (e.g., kanamycin resistance) Onceisolated, the full length vector plasmid is digested with Pacd torelease the Ad derived genome sequences from the bacterial plasmidsequences, and introduced directly into the appropriatetrans-complementing mammalian cell line to begin propagation of thetransgene encoding vector.

As originally described by He et al, the pAdEasy system can onlygenerate [E1-, E3-, and/or E4-] Ad vectors. We improved the method of Heet al. in order to allow for the construction of Ad vectors deleted notonly for E1, E3 and/or E4 genes but also for the polymerase, pTP, 100K,and/or the IVa2 genes.

To facilitate the cloning of deletions we had already generated in Ad5into pAdEasy1, this plasmid was digested with BamHI to divide theplasmid roughly in half. The larger of the two Bam HI fragments wasallowed to re-ligate to itself to form pAdΔBamHI(Δ21696-33422). ThepAdΔBamHI(Δ21696-33422) plasmid contains pAdEasy1 derived sequencesexcept the AdEasy1 sequences 21696-33422. The smaller of the two BamHIfragments contains pAdEasy1 derived sequences 21696-33422 including theE3 deletion, and was cloned into the BamHI site of pcDNA3 (any bacterialplasmid containing BamHI sites and an antibiotic marker could beutilized at this step, pcDNA3 was utilized in this instance) to formpAd(21696-33422). The final daughter plasmids pAdΔBamHI(Δ21696-33422)and pAd(21696-33422) are used as templates for the introduction of otherdeletions into the Ad genome. Once a deletion has been introduced intoeither of the two plasmids, (see below) the pAdEasy derived sequencesfrom each plasmid are re-ligated (via BamHI restriction ezyme digestion,isolation of the appropriate subfragments of DNA, and ligated) to form anew parent pAd vector now containing all desired deletions in additionto the original E1 and E3 deletions present in the He et al. constructs.The multiply deleted pAd plasmid is capable of being recombined with anyof the shuttle plasmids described by He et al, to generate full Advector genomes that are deleted for E1,E3, as well as the polymerase,preterminal protein, IVa2, and/or 100K genes separately, orsimultaneously

EXAMPLE 31 Generation of the Polymerase Deletion within a Parent pAdPlasmid and Subsequent Vector Production

pAdΔBamHI(Δ21696-33422) was digested with MunI, and the subfragments ofDNA between nucleotides 4036-9158 were replaced with a homologoussubfragment of DNA derived from MunI partial digestion of pAdAscLΔpol(Example 2) to successfully produce polymerase deleted Ad vectors. Thepartial Muni DNA subfragment of pAdAscLΔpol contains the previouslydescribed deletion of the polymerase gene (nt. 7274-7882 of the Ad5genome). The resultant plasmid pAdΔBamHI(Δ21696-33422) Δpol was digestedwith BamHI, and the ˜12kb BamHI subfragment (encompassing Ad sequences21696-33422) after BamHI digestion of pAd(21696-33422) was ligateddirectionally into pAdΔBamHI(Δ21696-33422) Δpol to generate pAdΔpol.This plasmid has been recombined with shuttle plasmids (as described byHe et al., (1998) Proc. Natl. Acad. Sci. USA 95:2509) containing markergenes (e.g., bacterial β-galactosidase) as well as several versions ofthe human lysosomal α-glucosidase (GAA) gene (e.g., the 3.3, e.g.,h5′sGM sequence described in Example 23, and 3.8 kb, the GAA sequencefrom pcDNA3-GAA in Example 23, versions encoding the human GAAprecursor, without and with 5′ flanking sequences, as described in VanHove et al., (1996) Proc. Natl. Acad. Sci. USA 93:65) flanked by eitherthe CMV enhancer/promoter, or the Elongation factor 1-alphaenhancer/promoter. Homologous recombination between the pAdΔpol and therespective shuttle plasmids resulted in the successful isolation of fulllength multiply deleted [E1-,E3-,pol-]Ad vector genomes containing therespective transgenes. PacI digestion of the vector genome containingplasmids, followed by transfection into E1 and polymerase expressingcell lines (e.g., B6 or C7) resulted in the successful production andpropagation of the desired multiply deleted Ad vectors, only in therespectively transcomplementing cell lines.

EXAMPLE 32 Generation of the Polymerase and Preterminal ProteinDeletions within a Parent pAd Plasmid and Subsequent Vector Production

pAdΔBamHI(Δ21696-33422) Δpol, was digested with BspEI, effectivelycleaving the plasmid into three subfragments, the largest was isolatedand ligated to the 2050 bp BspEI subfragment of the plasmidpAdAscLΔpolΔpTP (also referred to a pAdAscLΔpp (1.6)). The 2050 bpsubfragment of pAdAscLΔpol, ΔpTP encompasses the pTP deletion (nt8631-9197). The resultant plasmid pAdΔBamHI(Δ21696-33422) Δpol,ΔpTP wasdigested with BamHI, and the ˜12kb BamHI subfragment (encompassing Adsequences 21696-33422) present after BamHI digestion of pAd(21696-33422)was ligated directionally into pAdΔBamHI(Δ21696-33422) Δpol, ΔpTP, togenerate pAdΔpol,ΔpTP. This plasmid has been recombined with shuttleplasmids (as described by He et al., (1998) Proc. Natl. Acad. Sci. USA95:2509) containing marker genes (e.g., bacterial β-galactosidase) aswell as several versions of the human lysosomal α-glucosidase (GAA) geneencoding the human GAA precursor, without and with 5′ flanking sequences(e.g., the 3.3 and 3.8 kb versions, for example, the h5′sGAA and GAAsequences in Example 23), as described in Van Hove et al., (1996) Proc.Natl. Acad. Sci. USA 93:65) flanked by either the CMV enhancer/promoter,or the Elongation factor 1-alpha enhancer/promoter. Homologousrecombination between the pAdΔpol, ΔpTP and the respective shuttleplasmids resulted in the successful isolation of full length multiplydeleted [E1-,E3-,pol-,pTP-]Ad vector genomes containing the respectivetransgenes. Pacd digestion of the vector genome containing plasmids,followed by transfection into E1, polymerase, and preterminal proteinexpressing cell lines (e.g., C7) resulted in the successful productionand propagation of the desired multiply deleted Ad vectors only in therespectively transcomplementing cell lines.

EXAMPLE 33 Generation of the IVa2, Polymerase, and Preterminal ProteinDeletions within a Parent pAd Plasmid

pAdΔBamHI(Δ21696-33422) Δpol, was digested with Muni, and thesubfragments of DNA between nucleotides 4036-9158 were replaced with ahomologous subfragment of DNA derived from Muni partial digestion ofpAdAscLΔIVa2, Δpol, ΔpTP (also called pAdAscLΔIV2, Δpp (1.6); FIG. 36)to successfully produce E1, polymerase, pTP, and IVa2 deleted Advectors. The partial MunI DNA subfragment of pAdAscLΔIV2, Δpol, ΔpTPcontains the previously described deletion of the polymerase gene (nt.7274-7882 of the Ad5 genome) the preterminal protein gene (nt 8631-9197of the AdS genome), and the IVa2 deletion (nt 48305766 of the Ad5genome). The resultant plasmid pAdΔBamHI(Δ21696-33422) ΔIVa2, Δpol,ΔpTPwas digested with BamHI, and the ˜12kb BamHI subfragment (encompassingAd sequences 21696-33422) released after BamHI digestion ofpAd(21696-33422) was ligated directionally into pAdΔBamHI(Δ21696-33422)ΔIVa2, Δpol, ΔpTP, to generate pAdΔIVa2, Δpol, ΔpTP. The vector [E1-,E3-, IVa2-, pol-, pTP-(1.6)]Ad is generated following the techniquesprovided herein or other techniques known in the art.

This plasmid is recombined with any of the previously described shuttleplasmids (containing for example marker genes, the GAA genes, or anyother DNA sequence) to generate the respectively deleted Ad vectors inthe appropriate trans-complementing cell lines.

Routine modifications of the techniques described in the precedingparagraphs are used to generate [E1-, E3-, IVa2-]Ad, [E1-, E3-, IVa2-,pol-]Ad, and [E1-, E3-, IVa2-, pol-, pTP-(2.4)]Ad from the shuttleplasmids pAdAscLΔIVa2 (FIG. 19), pAdAscLΔIV2, Δpol (FIG. 38), andpAdAscLΔIVa2, Δpp (2.4) (FIG. 37), respectively.

EXAMPLE 34 Generation of the 100K Deletion within a Parent pAd Plasmidand Subsequent Vector Production

pAdΔBamHI(21696-33422) a 17.1 kb plasmid containing Ad derived sequencethat also encompass the entire 100K gene, was digested with Nhe I togenerate three DNA subfragments. Nhe I restriction digestion removes a687 bp fragment within the 100K gene between sequences 24999 to 25686 ofthe Ad5 genome. An additional Nhe I site at sequence 31509 of the Ad5genome liberates a 3143 bp fragment as well as the remaining 13,302 bpfragment of the pAd(21696-33422) plasmid. Careful attention was given toensure that the 687 bp deletion does not interfere with other potentialreading frames that overlap 100K or lie on the opposite coding strand of100K. To generate pAd(21696-33422) Δ100K, the 13,302 bp Nhe I digestedpAd(21696-33422) subfragment was ligated to the 3143 bp Nhe I fragment.This plasmid was digested with BamHI, and the DNA subfragment wasligated in the correct orientation into BamHI digestedpAdΔBamHI(Δ21696-33422), to generate pAdΔ100K. The pAdΔ100K has beenrecombined with shuttle plasmids that contained marker genes (e.g., thebacterial B-galactosidase flanked by the CMV enhancer/promoter).

Homologous recombination between the pAdΔ100K and the respective shuttleplasmids resulted in the successful isolation of full length multiplydeleted [E1-, 100K-] Ad vector genomes containing the respectivetransgenes. Pacl digestion of the vector genome containing plasmids,followed by transfection into E1, and 100K expressing cell linesresulted in the successful production and propagation of the desired,multiply deleted Ad vectors. These viruses were found to have a 50 foldincreased ability to propagate and produce infectious transducing unitsin the E1 and 100K expressing cell lines, as compared to cells onlyexpressing the E1 genes, confirming that the 100K deletion is asignificant deletion that incapacitates vectors containing it.

This pAdΔ100K plasmid is recombined with any of the previously describedshuttle plasmids (containing for example marker genes, the GAA genes, orany other DNA sequence) to generate the respectively deleted Ad vectorsin the appropriate trans-complementing cell lines.

EXAMPLE 35 Generation of the IVa2, Polymerase, Preterminal Protein, and100K Deletions within a Parent pAd Plasmid for Subsequent VectorProduction

pAdΔBamHI(Δ21696-33422) ΔIVa2, Δpol, ΔpTP is digested with BamHI. Thepolymerase, preterminal protein, and IVa2 deletions are ligated into theBamHI subfragment of pAd(21696-33422) Δ100K encompassing the 100Kdeletion to generate pAdΔIVa2, Δpol, ΔpTP, Δ100K. The vector [E1-, E3-,IVa2-, pol-, pTP- (1.6), 100K-)]Ad is generated following the techniquesprovided herein or other techniques known in the art.

This plasmid is recombined with any of the previously described shuttleplasmids (containing for example marker genes, the GAA genes, or anyother DNA sequence) to generate the respectively deleted Ad vectors inthe appropriate trans-complementing cell lines.

EXAMPLE 36 Adh5′sGAAΔpol, Ad/EF1-α/hGAAΔpol, and Ad/EF1-α/h5′sGAAΔpol

Using routine modifications of the methods described in Example 30regarding production of adenovirus vectors using bacterial homologousrecombination AdhGAAΔpol (CMV promoter) Adh5′sGAAΔpol (CMV promoter),and Ad/EF1-α/hGAAΔpol (EF1-α promoter) have been generated. The 5′sGAAsequence is the sequence of pcDNA3-5′sGAA (Example 23), which lacks mostof the 5′ untranslated sequences of the hGAA gene. In addition, usingroutine modifications of the methods described herein,Ad/EF1-α/h5′sGAAΔpol (EF1-α and shorted hGAA gene) is generated.

These vectors are administered to mice or other subjects in vivo,including mammalian subjects, in particular, human subjects. Inparticular, these vectors are administered to subjects with GAAdeficiency to achieve a therapeutic effect (as described hereinabove).

In particular, these vectors are administered to wild-type or GAA-KOmice and tissue/plasma GAA activity and glycogen accumulation/depletionis monitored as described in Example 25.

EXAMPLE 37 AdhGAAΔpp, Adh5′sGAAΔpp, Ad/EF1-α/hGAAΔpp, andAd/EF1-α/h5′sGAAΔpp

Using routine modifications of the methods described in Example 30regarding the production of adenovirus vectors using bacterialhomologous recombination AdhGAAΔpp (CMV promoter) and Adh5′sGAAΔpp (CMVpromoter) have been generated. The Δpol, ΔpTP deletions are as describedin Example 3 for AdLacZΔpp (deletions from about nucleotides 7274 to7881 and from about nucleotides 9198 to 9630 of the Ad5 genome). The5′sGAA sequence is the sequence of pcDNA3-5′sGAA (Example 23), whichlacks most of the 5′ untranslated sequences of the hGAA gene. Inaddition, using routine modifications of the methods described herein,Ad/EF1-α/hGAAΔpp (EF1-α promoter) and Ad/EF1-α/h5′sGAAΔpol (EF1-α andshorted hGAA gene) are generated.

These vectors are administered to mice or other subjects in vivo,including mammalian subjects, in particular, human subjects. Inparticular, these vectors are administered to subjects with GAAdeficiency to achieve a therapeutic effect (as described hereinabove).

In particular, these vectors are administered to wild-type or GAA-KOmice and tissue/plasma GAA activity and glycogen accumulation/depletionis monitored as described in Example 25.

All patent publications cited in this specification are herebyincorporated by reference in their entirety as if each individualpublication was specifically and individually indicated to beincorporated by reference.

As will be apparent to those skilled in the art to which the inventionpertains, the present invention may be embodied in forms other thanthose specifically disclosed above without departing form the spirit oressential characteristics of the invention. The particular embodimentsof the invention described above are, therefore, to be considered asillustrative and not restrictive. The scope of the invention is as setforth in the appended claims rather than being limited to the examplescontained in the foregoing description.

2 1 20 DNA Artificial Sequence Description of Artificial Sequence Primer1 tccaggccat ctccaaccat 20 2 23 DNA Artificial Sequence Description ofArtificial Sequence Primer 2 tctcagtctc catcatcatc acg 23

That which is claimed is:
 1. A method of treating a subject with alysosomal acid α-glucosidase deficiency comprising administering abiologically-effective amount of a propagation-efective adenovirusencoding a lysosomal acid α-glucosidase to the liver of the subject,wherein the liver expresses and secretes the encoded lysosomal acidα-glucosidase, which is transported to a muscle tissue in atherapeutically-effective amount.
 2. The method of claim 1, wherein theadenovirus encodes a lysosomal acid α-glucosidase precursor protein. 3.The method of claim 1, wherein the adenovirus vector is selected fromthe group consisting of AdhGAAΔpol, Ad/EF1-α/hGAAΔpol, Adh5′sGAAΔpol,Ad/EF1-α/h5′sGAAΔpol, AdhGAAΔpp, Ad/EF1-α/hGAAΔpp, Adh5′sGAAΔpp,Ad/EF1-α/h5′sGAAΔpp.
 4. The method of claim 1, wherein the adenovirus isadministered to the liver by a method selected from the group consistingof intravenous administration, intraportal administration, intrabiliaryadministration, intra-arterial administration, and direct injection intothe liver parenchyma.
 5. The method of claim 1, wherein the subject is amammalian subject.
 6. The method of claim 5, wherein the subject is ahuman subject.
 7. The method of claim 1, wherein the adenovirus isadministered to the liver by intravenous administration.
 8. A method oftreating a subject with lysosomal acid α-glucosidase deficiency,comprising administering to the subject a therapeutically-effectiveamount of a propagation-defective adenovirus comprising an adenovirusgenome comprising (i) a heterologous nucleotide sequence that encodes alysosomal acid α-glucosidase, and (ii) one or more deletions in the 100Kregion, wherein the deletion(s) essentially prevents the expression of afunctional 100K protein from the deleted region.
 9. The method of claim8, wherein the lysosomal acid α-glucosidase is a human lysosomal acidα-glucosidase.
 10. The method of claim 8, wherein the subject isselected from the group consisting of avian subjects and mammaliansubjects.
 11. The method of claim 10, wherein the subject is a humansubject.
 12. The method of claim 8, wherein the adenovirus isadministered by a method selected from the group consisting oftransdermal, intravenous, subcutaneous, intradermal, intramuscular, andintraarticular administration.
 13. The method of claim 8, wherein theadenovirus is delivered to the liver by a method selected from the groupconsisting of intravenous administration, intraportal administration,intrabiliary administration, intra-arterial administration, and directinjection into the liver parenchyma.
 14. The method of claim 8, whereinthe adenovirus is administered by intravenous administration.
 15. Amethod of treating a subject with lysosomal acid α-glucosidasedeficiency, comprising administering to the subject atherapeutically-effective amount of a propagation-defective adenoviruscomprising an adenovirus genome comprising (i) a heterologous nucleotidesequence that encodes a lysosomal acid α-glucosidase, and (ii) one ormore deletions in the IVa2 region, wherein the deletion(s) essentiallyprevents the expression of a functional IVa2 protein from the deletedregion.
 16. The method of claim 15, wherein the lysosomal acidα-glucosidase is a human lysosomal acid α-glucosidase.
 17. The method ofclaim 15, wherein the subject is selected from the group consisting ofavian subjects and mammalian subjects.
 18. The method of claim 17,wherein the subject is a human subject.
 19. The method of claim 15,wherein the adenovirus is administered by a method selected from thegroup consisting of transdermal, intravenous, subcutaneous, intradermal,intramuscular, and intraarticular administration.
 20. The method ofclaim 15, wherein the adenovirus is delivered to the liver by a methodselected from the group consisting of intravenous administration,intraportal administration, intrabiliary administration, intra-arterialadministration, and direct injection into the liver parenchyma.
 21. Themethod of claim 15, wherein the adenovirus is administered byintravenous administration.
 22. A method of treating a subject withlysosomal acid α-glucosidase deficiency, comprising administering to thesubject a therapeutically-effective amount of a propagation-defectiveadenovirus comprising an adenovirus genome comprising (i) a heterologousnucleotide sequence that encodes a lysosomal acid α-glucosidase, and(ii) one or more deletions in the preterminal protein region, whereinthe deletion(s) essentially prevents the expression of a functionalpreterminal protein from the deleted region.
 23. The method of claim 22,wherein the adenovirus is selected from the group consisting ofAdhGAAΔpp, Ad/EF1-α/hGAAΔpp, Adh5′sGAAΔpp, and Ad/EF1-α/h5′sGAAΔpp. 24.The method of claim 22, wherein the lysosomal acid α-glucosidase is ahuman lysosomal acid α-glucosidase.
 25. The method of claim 22, whereinthe subject is selected from the group consisting of avian subjects andmammalian subjects.
 26. The method of claim 25, wherein the subject is ahuman subject.
 27. The method of claim 22, wherein the adenovirus isadministered by a method selected from the group consisting oftransdermal, intravenous, subcutaneous, intradermal, intramuscular, andintraarticular administration.
 28. The method of claim 22, wherein theadenovirus is delivered to the liver by a method selected from the groupconsisting of intravenous administration, intraportal administration,intrabiliary administration, intra-arterial administration, and directinjection into the liver parenchyma.
 29. The method of claim 22, whereinthe adenovirus is administered by intravenous administration.
 30. Amethod of treating a subject with lysosomal acid α-glucosidasedeficiency, comprising administering to the subject atherapeutically-effective amount of a propagation-effective adenoviruscomprising an adenovirus genome comprising (i) a heterologous nucleotidesequence that encodes a lysosomal acid α-glucosidase, and (ii) one ormore deletions in the adenovirus polymerase region, wherein thedeletion(s) essentially prevents the expression of a functionalpolymerase protein from the adenovirus genome.
 31. The method of claim30, wherein the adenovirus is selected from the group consisting ofAdhGAAΔpol, Ad/EF1-α/hGAAΔpol, Adh5′sGAAΔpol, Ad/EF1-α/h5′sGAAΔpol. 32.The method of claim 30, wherein the lysosomal acid α-glucosidase is ahuman lysosomal acid α-glucosidase.
 33. The method of claim 32, whereinthe subject is selected from the group consisting of avian subjects andmammalian subjects.
 34. The method of claim 30, wherein the subject is ahuman subject.
 35. The method of claim 30, wherein the adenovirus isadministered by a method selected from the group consisting oftransdermal, intravenous, subcutaneous, intradermal, intramuscular, andintraarticular administration.
 36. The method of claim 30, wherein theadenovirus is delivered to the liver by a method selected from the groupconsisting of intravenous administration, intraportal administration,intrabiliary administration, intra-arterial administration, and directinjection into the liver parenchyma.
 37. The method of claim 30, whereinthe adenovirus is administered by intravenous administration.